![Chapter 1
Isolation and Purification of Viable PGCs from Mouse
Embryos
Massimo De Felici
Abstract
In all organisms with sexual reproduction, sperm and oocytes derive from embryonic precursors termed
primordial germ cells (PGCs) which pass on genetic information to subsequent generations. Studies aimed
to unravel PGC development at molecular level in mammals can be traced at the early 1980s and were
hampered by the difficulty in obtaining both sufficient quantities and purity of PGCs. For many labora-
tories, the isolation and purification methods of PGCs at different stages from embryos are the most
shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. In
the present chapter, I focus on immunomagnetic cell sorting (MACS) and fluorescence-activated cell
sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to
12.5 dpc embryos before their differentiation in oogonia/oocytes in female and prospermatogonia in male.
Key words Primordial germ cells, MiniMACS, SSEA-1, CD-15, Gonadal ridges, AGM, FACS,
OCT4, eGFP
1 Introduction
In the mouse embryo, it is now well established that PGC precur-
sors are specified from a small group of proximal epiblast cells and
become identifiable as alkaline phosphatase (AP)- and PRDM1-
positive cells at the base of allantois in the extraembryonic meso-
derm at around 7.5 days post coitum (dpc) (for a review, see Ref.
[1]). It is not clear if at this time these cells are part of a heteroge-
neous population of pluripotent stem cells destinated to give rise to
various lineage (for a review, see Ref. [2]). In any case, during
gastrulation, PGCs can be traced within the developing hindgut
endoderm at 7.75 dpc and into the aorta-gonad-mesonephros
(AGM) region at 9.5–10.5 dpc. Between 11.5 and 12.5 dpc,
PGCs become germline determined and initiate their sexual differ-
entiation into prospermatogonia (male) and oogonia (female),
instructed by the surrounding microenvironment of the gonadal
Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology,
vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
3](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-18-320.jpg)
![niche. From about 13.5 dpc, prospermatogonia undergo a tempo-
rary GO mitotic arrest while oogonia enter the prophase of meiosis
I as primary oocytes.
4 Massimo De Felici
PGCs representing a key cell type for analysis of germ-soma
differentiation as well as pluripotency at the molecular level. More-
over, in the last years, the possibility to generate primordial germ
cells–like cells (PGCLCs) in vitro able to generate male and female
gametes from pluripotent stem cells of various mammalian species
including humans (for a review, see Ref. [3]) increased exponen-
tially the interest about such unique cell type. Today single-cell
genome and transcriptome sequencing methods enabling the
simultaneous molecular analysis of hundreds, or thousands of
cells, inside heterogeneous populations are considerably increasing
our capability to analyze the PGC genome and transcriptome (for
example, see Refs. [4–6]. Nevertheless, for many laboratories, the
isolation and purification methods of PGCs at different stages from
embryos remain the most shortcut and affordable tool to study
many aspects of their development at cellular and molecular levels.
Historically, the isolation and purification of PGCs from mouse
embryos between 8.5 and 12.5 dpc were attempted using cellular,
molecular, and transgenic approaches in parallel with the progress
of technologies as summarized in Table 1. At these stages, PGCs are
characterized by motility capability and active proliferation; they are
for the most part single or form small clusters and do not form firm
contacts with the surrounding extracellular matrix molecules and
surrounding somatic cells. Both female and male mouse PGCs
express surface markers such as tissue-nonspecific alkaline phospha-
tase (TNAP), the carbohydrate epitopes bearing terminal Lewis X
determinants (Stage-specific embryonic antigen-1 (SSEA-1) and
the antigenic determinant of monoclonal antibody EMA-1, E-cad-
herin, and the c-Kit and CXCR4 receptors. In principle, antibodies
against all these surface molecules can be used to purify PGCs at
these stages. Moreover, PGCs express several genes typical of plu-
ripotent stem cells such as Pou5f1 (Oct4), Sox2, Nanog and of the
germline such Nanos3, Dazl and Ddx4 (Vasa). Therefore, PGCs
can be purified also from embryos of transgenic mice carrying
reporter genes under the control of such genes (for example, see
Refs. [12, 13]).
In the present chapter, I focus on immunomagnetic cell sorting
(iMACS) and fluorescence-activated cell sorting (FACS) methods
used in my laboratory for the purification of mouse PGCs from
10.5 to 12.5 dpc embryos using anti-SSEA-1 (CD15) MicroBeads
and the transgenic mouse line Oct4ΔPE: eGFP (OG2),
respectively.](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-19-320.jpg)
![Purification of Mouse PGCs 5
Table 1
Timetable of the methods used for the first time to isolate and purify mouse PGCs
Heath J. K. (1978) [7] (Cell 15, 299–306) Manual dissection of gonadal ridges
De Felici M. and McLaren A. (1982) [8] Percoll gradient
McCarrey J. R. et al. (1987) [9] Immunopurification by FACS
Massimo De Felici and Maurizio Pesce (1995) [10] Immunoaffinity adhesion
Pesce, M., and De Felici, M. (1995) [11] Immunopurification by MACS
Abe K. et al. (1996) [12] (Dev. Biol. 189, 468–472) Transgenic TNAPβ-geo
PGCs by FACS-gal
Szabò P. E. et al. (2002) [13] Transgenic Oct4ΔPE:eGFP PGCs by FACS
Mayanagi T. et al. (2003) [14] Nicodenz gradient
2 Materials and Instruments
2.1 Isolation of PGCs
by MiniMACS
1. 10.5–12.5 dpc pregnant females of desired genetic background
(see Note 1).
2. 1xPBS, sterile, for dissecting.
3. Hanks’ Balanced Salt Solution (HBSS) or M2 (Sigma-Aldrich).
4. Trypsin/EDTA solution (0.025% trypsin and 0.01% EDTA) in
PBS (Gibco).
5. DNase (1 mg/mL) solution (STEMCELL Technology).
6. Culture-tested fetal calf serum (FCS).
7. Poly-L-lysine-coated glass coverslips: soak coverslips in
200 μg/mL poly-L-lysine in distilled water for at least 1 h at
r. t., wash three times in distilled water and air-dry.
8. Chromogenic solution for alkaline phosphatase
(AP) detection: immediately before use dissolve 1 mg/mL
fast red TR salt or fast blue BB salt (Sigma-Aldrich) in distilled
water and add 40 μL/mL naphthol AS-BI or AS-MX alkaline
solution (Sigma-Aldrich).
9. Fine scissors, dissection forceps and No. 5 watchmaker’s for-
ceps, sterile.
10. Eppendorf or equivalent single channel 10–100 μL pipette.
11. 18- and 23-gauge needle and 1 mL syringe for tissues
dissection.
12. 5 mL test tubes and 1.5 and 2 mL Eppendorf tubes.
13. Stereomicroscope (the author uses a Wild M5a from Leica,
maximum magnification 100×, equipped with both incident
and transmitted light sources, the latter preferably with a mov-
able mirror).](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-20-320.jpg)
![6 Massimo De Felici
14. Anti-SSEA-1 (CD15) MicroBeads (130-094-530, Miltenyi
Biotech).
15. MACS buffer: phosphate-buffered saline (DPBS) without
Ca2
+ and Mg2
+, with 0.5% bovine serum albumin (BSA), and
2 mM EDTA (keep buffer cold at 2-8 °C and degas before
use, as air bubbles could block the column).
16. MiniMACS™ Separator (Miltenyi Biotech).
17. MACS MultiStand (Miltenyi Biotech).
18. MACS MS separation columns (Miltenyi Biotech).
19. Class II Biological Hazard laminar flow hood (see Note 2).
2.2 Isolation of PGCs
from OG2 Mice by
FACS
Materials listed in the previous section from 2 to 11 are required
also in this procedure.
1. 10.5–12.5 dpc pregnant CD1 females previously mated with
OG2 homozygous males (see Note 3).
2. Hanks’ Balanced Salt Solution (HBSS) or M2 (Sigma-Aldrich)
plus 1 mg/mL BSA.
3. 5 mL FACS tubes compatible with flow cytometer.
4. Sorting flow cytometer (see Note 4).
3 Methods
3.1 Dissection of
AGM and GRs
At 10.5 dpc, the region dissected away from the embryo (hindgut,
dorsal mesentery, and gonadal ridges) corresponds to the region
termed AGM (aorta, gonad, mesonephros) containing the most
part of migratory PGCs (about 1000/embryo, while at 11.5 and
12.5 dpc PGCs are obtained from the gonadal ridges (GRs, about
3000/and 8000/embryo) ([15], and our unpublished data).
Embryonic sex can be determined only by PCR of genomic DNA
using specific primers for 10.5 and 11.5 dpc samples (see Note 5) or
by assessing gonad morphology for 12.5 dpc (Fig. 1).
The procedure described in the following steps is routinely
used in the author’s laboratory, but it can be varied according to
the operator’s preference and experience.
Steps
1. Lay the freshly killed animal on its back and soak the abdomen
with 70% alcohol. CD1 mice, usually employed in the author’s
laboratory, were housed and mated under conventional condi-
tions (see Note 1). Positive plug test is considered 0.5-day post
coitum. The developmental stage of embryos can be precisely](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-21-320.jpg)
![8 Massimo De Felici
dorsal mesentery and the hindgut. Peel away these and asso-
ciated GRs (gonad + mesonephros) from the dorsal region of
the embryo and transfer them into HBSS or M2 (Fig. 2).
10. For 11.5 and 12.5 dpc embryos, scoop out liver and any
remnants of intestine with No. 5 watchmaker’s forceps and
identify the GRs lying on the dorsal wall of the embryos on
either side of the abdominal aorta. Slide the tips of the
No. 5 watchmaker’s forceps behind each GR and remove
them from the embryo.
11. Transfer the GRs to HBSS or M2 and separate the gonads from
mesonephroi using 25G needles.
3.2 Isolation of 10.5–
12.5 dpc PGCs by
MiniMACS
In this method, PGCs are separated from the other cell types by
positive selection. This means that they are labeled with superpar-
amagnetic microbeads conjugated to highly specific antibodies.
During separation, the magnetically labeled PGCs are retained
within a column, while unlabeled cells flow through. After a wash-
ing step, the column is removed from the magnetic field of the
separator, and PGCs are eluted. Positive selection can be performed
by direct or indirect magnetic labeling. The following procedure
describes a direct labelling of PGCs by anti-SSEA-1 MicroBeads
purchased from Miltenyi Biotechnology.
The simplicity and low cost make the MiniMACS separation
procedure the method of choice for rapidly obtaining reasonable
numbers of purified germ cells and somatic cells that can be used
immediately for biochemical studies or be grown in suitable in vitro
culture systems. In 2–3 h of work, one person should be able to
isolate from 10 embryos of 10.5, 11.5, and 12.5 dpc (a reasonable
yield from a pregnant CD1 female) about 5000–7000 (purity
70–80%), 10,000–15,000, and 40,000–50,000 PGCs (purity
85–95%), respectively (Fig. 3). The presence of supermagnetic
microbeads coated with antibodies on the cell surface might be
harmful for certain cell functions. However, we cultured PGC
purified by MiniMACS for several days without any apparent dele-
terious effect (for example see Ref. [16]).
Steps
1. 10.5 dpc AGM (usually 10–20) or 11.5–12.5 gonads (usually
20–40) are transferred with 100 μL Eppendorf pipette in mini-
mal amount of medium into a 1.5 mL Eppendorf tube contain-
ing 0.5 mL of trypsin-EDTA.
2. After about 5 min incubation at 37 °C carefully remove the
solution and wash the tissues twice with 1 mL of HBSS or
M2 + 5% FCS, allowing them to settle on the bottom of the
tube. Leave the tissue in about 100 μL of medium and pipette
up and down with the Eppendorf pipette for their complete](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-23-320.jpg)
![10 Massimo De Felici
Fig. 4 A MiniMACS separation unit form Miltenyi Biotec
8. To assess PGC purity, a small sample of the cell suspension
(20–30 μL) can be transferred onto a polylysinated coverglass
and stained for AP.
3.3 Isolation of 10.5–
12.5 dpc PGCs by FACS
At these developmental stages, FACS can be used to purify viable
PGCs labeled with antibodies anti-SSEA-1 (CD15) or other sur-
face markers (e.g., EMA-1, E-cadherin, Kit) conjugated with vari-
ous fluorochromes commercially available from several companies.
Alternatively, PGCs can be isolated from transgenic mice expressing
reporter genes under the control of early PGCs genes (e.g., Pou5f1,
Prdm1, and Dppa3). Here we describe PGC purification from OG2
transgenic mice (Oct4ΔPE:eGFP) expressing enhanced green fluo-
rescent protein (eGFP) under the control of the Pouf1 (Oct4),
promoter and distal enhancer [17] (Fig. 5a).
Steps
1. After steps 1 and 2 of Subheading 3.2 Isolation of PGCs by
MiniMACS, add 1 mL of HBSS plus 1 mg/mL BSA contain-
ing 10 μg/mL DNase, centrifuge at 500 ×g for 10 min at 4 °C.
Remove supernatant, and resuspend the pellet in 0.5 mL of
HBSS with the Eppendorf pipette (see Note 6).
2. Transfer the cell suspension into a FACS tube on ice and
proceed to flow cytometer.
3. Cell debris and dead cells are excluded from the analysis based
on scatter signals and propidium iodide fluorescence. Viable
cells are gated based on forward scatter (FSC) vs. side scatter
(SSC) profile. eGFP vs. FSC will delineate eGFP-positive PGC](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-25-320.jpg)
![)
12 Massimo De Felici
4 Notes
1. Use the mouse strain of your choice, although we have fond
that outbred strains such as MF1 and CD1 mice give larger
numbers of PGCs than some inbred strains (e.g., BalbC). The
procedure described here and the mean number of PGCs
obtained are from CD1 embryos. Moreover, consider that
PGC number can vary considerably among embryos.
2. The safety cabinet is necessary to carry out the whole procedure
if sterility must be maintained.
3. The OG2 mice can be purchased from Jackson Laboratory, Cat
# 003715. To ensure large litter sizes and large numbers of
germ cells per gonad homozygous OG2 male can be mate with
CD1 females. For the present protocol, usually, a minimum of
two litters is pooled.
4. For the present procedure, cell sorting was performed on a
FACSAria II (Becton Dickinson) interfaced with the FACS-
DiVa 6.0 Software.
5. If sexing is required, each embryo is dissected into a 24-well
plate containing warm DMEM+10% FCS and a tail tip is taken
for PCR sexing. We used genomic PCR of mouse Ube1 [18] or
Xlr and Sly [19] genes. Once results are available (2–3 h
embryos are pooled based on sex.
6. For optimal performance, it is important to obtain a single-cell
suspension. Check a small sample of the cell suspension under a
microscope and if necessary, pass the suspension through a
23-gauge needle once more or filter through a 40 μm cell
strainer (BD Falcon). The percent of PGCs in the cell suspen-
sion estimated by APase staining (see step 8) was less than 0.1%
and approximately 5% and 30–35% at 10.5, 11.5, and 12.5 dpc,
respectively.
7. Although the most part of PGCs showed high eGFP, we and
others observed a little population of weakly eGFP positive
cells. At 10.5 and 11.5 dpc, these latter represented between
9% and 12% of the whole eGFP positive cells. Hypotheses
about such minority PGC population are discussed in
[20]. Although FACS is the method of choice for cell sorting
when high cell purity is needed, it suffers, however, from
(sometimes very) high losses, requires trained personnel, and
is expensive to own and operate. The estimation of PGC recov-
ery under the present conditions varied from 65% to 70%. This
means that from 10 embryos of 10.5, 11.5, and 12.5 dpc
heterozygotes CD1/OG2 females, about 5000, 15,000, and
35,000 PGCs can be obtained, respectively. Finally, PGCs](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-27-320.jpg)
![Chapter 2
Purification and Transfection Methods of Chicken
Primordial Germ Cells
Luiza Chojnacka-Puchta and Dorota Sawicka
Abstract
Primordial germ cells (PGCs) play a special role in the vertebrate life cycle since they are the precursors of
germ cells through which the genome is passed to the next generations. PGCs are found in different
locations and variable numbers in the chick embryo, as in other species, depending on the developmental
stages. Here, we describe in detail a method based on the Percoll gradient, routinely used in our laboratory,
allowing us to obtain from blood and gonad anlages significant numbers of viable PGCs which can be
successfully cultured or efficiently genetically modified.
Key words Chicken primordial germ cells, Percoll density gradient, Electroporation, Lipofection,
Immuno-cyto-staining
1 Introduction
Primordial germ cells (PGCs) play a special role in the vertebrate
life cycle since they are the precursors of ova and spermatozoa
through which the genome is passed to the next generations.
In birds the germline is established early in the embryo, likely
by maternal specification [1]. In the chick embryo, PGC precursors
are initially detected as a scattered pattern in the area of pellucida,
the central region of the Eyal-Giladi and Kochav stage X embryo
[2]. From this position, these cells are translocated to the anterior
extraembryonic region called the germinal crescent, where they,
now termed PGCs, are incorporated into the forming extraembry-
onic vascular network [stages 8–10 according to Hamburger and
Hamilton (HH 8–10)] [3], and start to circulate within the blood-
stream (stage HH 11). Reaching the gonadal anlage region, PGCs
exit the blood vessels and colonize the gonads (stage HH 16).
About 30 PGCs are found at X stage embryo in the area of pellu-
cida, 200–250 in the germinal crescent, and more than 1000 are in
the gonads after embryo incubation for 7 days (stages 26–29 HH)
Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology,
vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
15](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-29-320.jpg)
![[5]. Considering such dynamics localization, at the 10 HH stage,
PGCs can be obtained from blastoderm, at 13–17 HH stages (2.5-
to 3-day-old embryos) from blood and at stages 26–29 HH stages
(5.5- to 6-day-old embryos) directly from the embryonic gonads.
PGCs isolated from these stages have been successfully used for
transfection [4, 5] or cryoconservation [6].
16 Luiza Chojnacka-Puchta and Dorota Sawicka
Purification methods for chick PGCs range from density gra-
dients using Nycodenz [7], Ficoll [8], or Percoll [9] to immuno-
magnetic cell sorting (MACS) and fluorescence-activated cell
sorting (FACS) [11]. Chick PGCs were purified from blood using
ammonium chloride-potassium (ACK) lysis buffer to eliminate red
blood cells, followed by in vitro culture [10]. Using such methods,
the purity of PGCs overcame 90% at best.
Chick PGCs can be identified based on morphological criteria,
i.e., large size and presence of a large amount of glycogen in the
cytoplasm [12]. Glycogen granules can be evidenced also by peri-
odic acid-Schiff (PAS) staining [13, 14]. Moreover, chick PGCs
were stained for alkaline phosphatase activity, a classical PGC
marker also in mammalian species [15], and for many immunolog-
ical epitopes for surface glycoproteins. The best known and char-
acterized is SSEA-1 (Stage-specific Embryonic Antigen-1) [16],
which is effective only after stage 10 HH. Other immunological
markers are EMA-1 (Epithelial Membrane Antigen) [17] expressed
from the crescent stage [18] and MVH (Mouse Vasa Homolog)
[19]. Interestingly, all these immunological markers are common
to mammalian PGCs (see Chap. 1, by M. De Felici, in the
present book)
Here we describe a simple and rapid method based on Percoll
density gradient suitable to purify chicken PGC from blood and
early embryonic gonads that can be successfully transfected using
the electroporation/lipofection method for germline manipula-
tion. Details are in Table 1.
Table 1
Average numbers of chicken embryos, PGCs isolated from blood and gonads, purity and efficiency of
transfection methods
Type
of
PGCs
Number
of
embryosa
Number of total
cells after
isolation
Number of total
after Percoll
purification
Purity of
PGCs
(%)
Electroporation
efficiency (%)
Lipofection
efficiency
(%)
bPGCs 60 11.6 × 106
1.76 × 106
64 75 50
gPGCs 30 4.53 × 106
7.56 × 105
N/A 73 39
Data obtained in our experiments
a
The number of embryos used to isolate PGCs depends on the quality of embryos and operator skills. Some of them can
be unfertilized or undeveloped](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-30-320.jpg)
![Chapter 3
Isolation and In Vitro Propagation of Human
Spermatogonial Stem Cells (SSCs)
Janmejay Hingu, Guillermo Galdon, Nicholas A. Deebel,
and Hooman Sadri-Ardekani
Abstract
Preservation of human spermatogonial stem cells (SSCs) may be suitable for young male patients at risk of
male infertility due to various causes, such as gonadotoxic treatment or genetic diseases. With optimal
cryopreservation, cell viability can be retained to reestablish spermatogenesis in the future through autolo-
gous transplantation or in vitro differentiation of SSCs. This protocol outlines techniques to optimize the
SSCs isolation and in vitro culture. With particular emphasis on the microscopic characteristics encoun-
tered, this protocol outlines a broader approach to processing tissues with varying morphologies among
patients.
Key words Male infertility, Spermatogonial stem cell (SSC), Isolation, Propagation
1 Introduction
Infertility is the inability to conceive after 12 months of unpro-
tected intercourse and remains a public health issue for both men
and women with equal prevalence. While many etiologies contrib-
ute to an infertility diagnosis, the male factor is present in approxi-
mately 50% of infertile couples and is the sole causative factor in
20–30% of couples [1]. Azoospermia (absence of sperm on two
consecutive centrifuged semen analyses) is the most severe form of
male infertility, ultimately leaving couples with limited options for
biological paternity in the setting of a negative microdissection
testicular sperm extraction procedure (mTESE) [2]. Despite a
negative sperm retrieval, testicular biopsies showing hyposperma-
togenesis, maturation arrest, or Sertoli cell only (SCO) may still
present putative spermatogonial stem cells (SSCs). Similarly, pre-
pubertal patients haven’t started spermatogenesis and cannot pro-
vide sperm yet. When faced with gonadotoxic treatment for
childhood malignancy, such as chemotherapy and radiation, they
Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology,
vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_3,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
27](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-40-320.jpg)
![may become azoospermic [3–5]. Consequently, current research
efforts have focused on SSC therapy to treat male factor infertility.
28 Janmejay Hingu et al.
Spermatogenesis begins with the SSC, a diploid cell that main-
tains the ability for self-renewal and downstream differentiation
into spermatozoa. Given its origin in spermatogenesis, using SSC
for male infertility is a logical choice for patients with maturation
arrest or initially diagnosed SCO with salvageable SSC on the
biopsy [2, 6]. Investigative SSC therapies have included SSC trans-
plantation and in vitro spermatogenesis. The formed differentiated
germ cells could potentially be used for in vitro fertilization (IVF),
either intracytoplasmic sperm injection (ICSI) or experimental
round spermatid injection (ROSI) [7–10]. Given that SSC is a
scarce population in azoospermic patients, in vitro propagation of
SSC is a critical step toward the feasibility of stem cell-based fertility
treatments.
This chapter aims to provide our institution’s protocol and
technical considerations for SSC culture in mammalians. Previous
works from our lab have included SSC isolation, propagation, and
partial differentiation to the round spermatid level in a unique
culture system [10–12]. This was initially performed in adult men
where SSC could be isolated and propagated for 28 weeks up to a
concentration of 18,540-fold [11]. Subsequently, this application
was expanded to prepubertal boys in which SSCs were cultured for
up to 29 weeks [12]. Most recently, this culture system has been
successfully applied to a mouse model and human patients with
Klinefelter syndrome, given the frequency of azoospermia, negative
mTESE, and the need for alternative therapies [8, 9, 13].
2 Materials
2.1 Reagents 1. Phosphate-buffered saline (PBS, 1×).
2. Minimal Essential Media 1×.
3. Fetal calf serum.
4. DNase (Roche) stock solution of 2 mg/mL. It is stored at -
20 °C in 200 μL aliquots. Keep in ice before use.
5. Collagenase NB 4 (Serva) stock solution of 10 PZ U/mL. It is
stored at -20 °C in 200 μL aliquots. Keep in ice before use.
6. Neutral Protease NB (Serva) stock solution of 10 DMC
U/mL. It is stored at -20 °C in 100 μL aliquots. Keep in ice
before use.
7. Enzyme mix: 5 mL 1× MEM DNase (8 μg/mL) + SERVA
Collagenase 4 PZ U + neutral Protease* 0.2 DMC
U + 20 μL extra DNase (2 mg/mL). If cells are intended for
transplantation into humans, Neutral Protease NB GMP Grade
is recommended.](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-41-320.jpg)
![Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells 35
3. Centrifuge cells (5 min, 180 g, with break). Remove the super-
natant and resuspend the pellet in 750 μL 1× MEM/20% FCS
(Room Temperature).
4. Transfer the cell suspension into a cryovial.
5. Add 750 μL 1× MEM/20% FCS/16% DMSO while gently
shaking the cryovial.
6. Transfer the cryovial into a cold Nalgene freezing box
(Mr. Frosty). Maintain at -80 °C for around 12 h but no
longer than 24 h.
7. Transfer the cryovial to a liquid N2 tank.
4 Notes
1. The reagents shown in Table 1 are added to 500 mL
StemPro™-34 serum-free medium to supplement the
medium. Final concentrations are based on the 500 mL
StemPro™-34 SFM, not the total volume.
2. After mixing reagents, filter the mixture with vacuum pressure
over a 22 μM filter. Immediately before use, add growth factors
shown in Table 2 to 50 mL supplemented StemPro™ SFM.
3. An aggressive change in the temperature of the cryovial may
trigger an expansive reaction of the gas inside. This may break
the cryovials, compromising the sample integrity or even
becoming a safety hazard for the operator. Ensure the initial
thawing is gradual and that appropriate safety guideline is
followed. We also recommend briefly opening the cryovials to
release air pressure as soon as the thawing process allows.
4. As previously reported, dimethyl sulfoxide DMSO is one of the
most commonly used cryoprotectants for testicular tissue [14],
but at room temperature, DMSO becomes cytotoxic. Three
time-sensitive consecutive washes are done to minimize tissue
exposure to DMSO at room temperature.
5. During this step, it is recommended to weigh the tissue to
calculate the isolation efficacy (number of cells per mg tissue).
6. Seminiferous tubules from simple testicular biopsies are usually
more laborious to separate than in mTESE samples. The
further separated the tubules are, the more adherent
(mucous-like) they appear and the more influential the enzy-
matic digestion will be.
7. The tubular cell layer should disintegrate at this point, releasing
the stem cells. The thickness of the tubular cell layer can differ
between patients. The incubation with enzymes can be
continued for longer to achieve the desired characteristics of
the peritubular cell layer. If cell clumps form, dissociate them
with trypsin-EDTA.](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-48-320.jpg)
![Chapter 4
Purification by STA-PUT Technique of Male Germ Cells
from Single Mouse and RNA-Extraction for Transcriptomic
Analysis
Chiara Naro, Claudio Sette, and Raffaele Geremia
Abstract
Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcrip-
tional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression
of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-
meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension
isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected
fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent
RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and
transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced
fertility poses to the obtaining of experimental animals.
Key words Male germ cells, Cell purification, STA-PUT, Gravimetric sedimentation, RNA extraction,
Transcriptome analysis
1 Introduction
Spermatogenesis is the complex differentiation process taking place
in the testes, leading to the generation of mature male gametes
[1, 2]. During this process spermatogonial stem cells undergo
profound genomic and morphological variations, which occur
along three subsequential phases: (i) amplification of spermatogo-
nia population, (ii) meiotic commitment and division into primary
and secondary spermatocytes, (iii) differentiation from haploid
round spermatids to mature spermatozoa (i.e. spermiogenesis)
[1, 2]. Initiation and accomplishment of each of these phases of
spermatogenesis is sustained by stage-specific patterns of gene
expression, providing each phase of the necessary protein repertoire
[3–7]. Understanding of the biological processes and of underlying
molecular mechanisms driving spermatogenesis relies on the
Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology,
vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_4,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
37](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-50-320.jpg)
![possibility to isolate and analyze male germ cells at the defined stage
of differentiation. In this regard, the STA-PUT technique repre-
sents, since the late 1960s, a pivotal procedure in this field of study.
This technique takes advantage of the fact that the individual types
of testicular cells have different size, thus sedimenting at different
velocities in a BSA gradient, independently from the shape. The
physical characteristics of the method have been extensively
described by Miller and Phillips using blood cells [8] and then
used for the separation of mouse male germ cells in the laboratory
of Dr. W.R. Bruce [9]. The effectiveness of the technique has been
re-evaluated from a morphological point of view [10], re-examined
also recently proposing different equipment [11], and widely used
for the study of DNA synthesis and transcriptional and translational
activity in the different phases of spermatogenesis and for a variety
of biochemical and metabolic studies [12–16]. The recent advance-
ment of transcriptomic approaches has provided remarkable
insights into the transcriptional and post-transcriptional molecular
mechanisms underlying the tightly and dynamically controlled gene
programs promoting male germ cells differentiation [17, 18]. Most
of these studies exploited RNA-sequencing (RNA-seq) from either
bulk-testis, collected at different time-points during the first syn-
chronous wave of murine spermatogenesis, or from male germ cells
at different stage of differentiation, isolated by either centrifugal
elutriation or Fluorescence-activated Cell Sorting (FACS) [3, 6,
7]. Both these approaches show limitations: i. presence of hetero-
geneous populations of somatic cells (i.e., Sertoli cells, Leydig cells)
is a confounding element in the identification of germ-cell-specific
gene expression patterns by RNA-seq of bulk-testes; ii. stage-
specific male germ cell isolation by centrifugal elutriation requires
injection of a high number of testicular cells obtained by pooling
testes from multiple mice. This latter aspect is a critical limitation
for transcriptomic studies aimed at the identification of gene
expression alterations correlated to spermatogenic defects in mice
with reduced reproductive potential, due to the small number of
animals. In our laboratory, we showed that gravimetric decantation
(STA-PUT) of testicular cellular suspension is a reliable method to
collect homogenous population of meiotic (spermatocytes) and
post-meiotic (round spermatids) male germ cells from a single
mouse, from which it is possible to isolate a sufficient amount of
RNA for reliable total-RNA seq analysis [4]. Moreover, we showed
this method to be applicable and reliable also for the isolation of
germ cells and subsequent transcriptomic analysis from a mouse
model with relevant spermatogenic defects, with reduced testis size
and impaired male germ cells differentiation, such as the Sam68
knock-out model [19, 20], therefore proving its applicability to the
late-generation transcriptomic analysis. Herein, we will describe a
detailed protocol for the fractionation of meiotic spermatocytes
and post-meiotic round spermatids from a single adult mouse and
the isolation of good-quality RNA for further transcriptomic
analysis.
38 Chiara Naro et al.](https://image.slidesharecdn.com/27770932-250520155121-b7df3112/85/Germ-Cell-Development-Methods-And-Protocols-1st-Edition-Marco-Barchi-51-320.jpg)
Germ Cell Development Methods And Protocols 1st Edition Marco Barchi
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- 5. GermCell Development Marco Barchi · Massimo De Felici Editors Methods and Protocols Methods in Molecular Biology 2770
- 6. M E T H O D S I N M O L E C U L A R B I O L O G Y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651
- 7. For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.
- 8. Germ Cell Development Methods and Protocols Edited by Marco Barchi Department of Biomedicine and Prevention, Section of Human Anatomy, Faculty of Medicine and Surgery, “Tor Vergata“ University of Rome, Rome, Italy Massimo De Felici Department of Biomedicine and Prevention, Section of Histology and Embryology, Faculty of Medicine and Surgery, “Tor Vergata” University of Rome, Rome, Italy
- 9. Editors Marco Barchi Department of Biomedicine and Prevention, Section of Human Anatomy, Faculty of Medicine and Surgery “Tor Vergata“ University of Rome Rome, Italy Massimo De Felici Department of Biomedicine and Prevention, Section of Histology and Embryology, Faculty of Medicine and Surgery “Tor Vergata” University of Rome Rome, Italy ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3697-8 ISBN 978-1-0716-3698-5 (eBook) https://doi.org/10.1007/978-1-0716-3698-5 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Paper in this product is recyclable.
- 10. Preface In sexually reproducing organisms, germ cells (GSs) bear two major responsibilities: the maintenance of the species through the transmission of hereditary traits, and species evolu- tion through the reshuffling of the genome. During early embryonal development, GCs develop from staminal GC precursors that give rise to primordial GCs (PGCs). The latter, by replicating and migrating, reach the primitive gonads where they will develop into male or female gamete precursors (spermatogonia or oogonia, respectively) depending on the assortment of the sex chromosomes complement (XY or XX, respectively) of the somatic gonadal cells. To reconstitute the diploid chromosome number at each fertilization, GCs undergo meiosis. The functional role of meiosis is to reduce the genome complement by half, which is accomplished by sequentially executing one round of DNA replication followed by two rounds of chromosome segregation. Between DNA replication and the first meiotic division in most organisms, paternal and maternal (homologous) chromosomes undergo homologous recombination. The latter is key for the reshuffling of the genome and proper segregation of homologous chromosomes at anaphase I. Importantly, recombina- tion is initiated by a programmed wave of double strand breaks (DSBs) that occurs along chromosomes in specific regions called “hotspots.” Precise positioning of hotspots is essential for successful recombination; therefore, alteration of the mechanisms determining hotspots may cause defects in pairing and synapsis of the homologous chromosomes, which may lead to sterility or to unbalanced segregation of chromosomes at anaphase I. In the latter case, when it is compatible with life (mostly it is the case of sex chromosomes missegregation), it causes in humans the onset of syndromes, such as Klinefelter (47, XXY), in which genetic-driven phenotypic manifestations are being studied. Thus, GCs are unique in their ability to transfer genetic information across generations. As such, proper understanding of the mechanisms underpinning their origin, regulation, and differentiation is key to understanding alterations of these processes, which are para- mount to the health of organisms and the survival of species. Until recent years, germ cell research was limited by the lack of in vitro models recapitulating male or female germ cell development. However, more recently, research in the field of stem cell biology has allowed an impressive acceleration in the expansion of new techniques for in vitro reconstruction of spermatogenesis and oogenesis, both in animal models and humans. In addition, the development of somatic cell reprogramming techniques makes it possible today to obtain stem cells from patient-derived somatic cells, providing a tool for molecular studies of human diseases, including the syndromes resulting from defects of developing germ cells. These advances promise to provide new insight into the understanding of basic biological aspects of germ cell biology, as well as the opportunity for in vitro manipulation of germ cells for toxicology studies and for genetic intervention where ethically appropriate. In this volume, we have collected well-established protocols for the isolation, purifica- tion, and establishment of in vitro GC systems at different stages of development in different organisms, including chickens, mice, rats, and humans. In addition, we describe cutting- edge analytic and informatic tools to study germ cell development at the single cell level and meiotic recombination. The volume is divided into four sections. The first section is devoted to methods for the isolation, purification, and transfection of PGCs, as well as methods for the purification of GCs from the fetal human testes and the adult testes of mice. Section II is v
- 11. divided into two parts. systems for induction an of the mouse germline mammalian ovaries and cells into pluripotent s section focuses on descr recombination initiation bination events occurrin The first part describes cytological methods for establishing in vitro d culturing of PGCs from PGC-like cells and induction and editing . The second part is dedicated to the isolation and culture of oocytes. Section III describes a protocol for reprogramming somatic tem cells (iPS) from patients with Klinefelter syndrome. The last iptions of bioinformatic pipelines for studying GC development and and a tool for image analysis of chromosome structure and recom- g in prophase I of meiosis. vi Preface The editors thank the many authors of these chapters for their willingness to share their protocols and expertise. Rome, Italy Marco Barchi Massimo De Felici
- 12. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I ISOLATION AND PURIFICATION OF PRIMORDIAL (AND) GERM CELLS 1 Isolation and Purification of Viable PGCs from Mouse Embryos. . . . . . . . . . . . . . 3 Massimo De Felici 2 Purification and Transfection Methods of Chicken Primordial Germ Cells . . . . . 15 Luiza Chojnacka-Puchta and Dorota Sawicka 3 Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells (SSCs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Janmejay Hingu, Guillermo Galdon, Nicholas A. Deebel, and Hooman Sadri-Ardekani 4 Purification by STA-PUT Technique of Male Germ Cells from Single Mouse and RNA-Extraction for Transcriptomic Analysis. . . . . . . . . . . . . . . . . . . . . 37 Chiara Naro, Claudio Sette, and Raffaele Geremia 5 Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Mark E. Gill, Hubertus Kohler, and Antoine H. F. M. Peters 6 Isolation and In Vitro Culture of Germ Cells and Sertoli Cells from Human Fetal Testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Celine M. Roelse, Arend W. Overeem, Yolanda W. Chang, Meriam Boubakri, and Susana M. Chuva de Sousa Lopes PART II ESTABLISHING IN VITRO SYSTEMS FOR STUDYING PRIMORDIAL (AND) GERM CELLS 7 Human Primordial Germ Cell-Like Cell Induction from Pluripotent Stem Cells by SOX17 and PRDM1 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Naoko Irie, Toshihiro Kobayashi, and M. Azim Surani 8 Induction of Primordial Germ Cell-Like Cells from Rat Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Mami Oikawa, Masumi Hirabayashi, and Toshihiro Kobayashi 9 Induction of Meiotic Initiation in Long-Term Mouse Spermatogonial Stem Cells Under Retinoid Acid and Nutrient Restriction Conditions. . . . . . . . . 113 Xiaoyu Zhang and Ning Wang 10 Lipofection-Based Delivery of CRISPR/Cas9 Ribonucleoprotein for Gene Editing in Male Germline Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Mariella Obermeier, Vera Rogiers, Tamara Vanhaecke, and Yoni Baert 11 Mouse In Vitro Spermatogenesis on 3D Bioprinted Scaffolds . . . . . . . . . . . . . . . . 135 Guillaume Richer, Tamara Vanhaecke, Vera Rogiers, Ellen Goossens, and Yoni Baert vii
- 13. viii Contents 12 Histological and Cytological Techniques to Study Perinatal Mouse Ovaries and Oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Nikoleta Nikou, Maria L opez Panadés, and Ignasi Roig 13 Method of Isolation and In Vitro Culture of Primordial Follicles in Bovine Animal Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Pritha Dey, Noemi Monferini, Ludovica Donadini, Valentina Lodde, Federica Franciosi, and Alberto Maria Luciano PART III SOMATIC REPROGRAMMING TO STUDYING HUMAN ANEUPLOIDY FEATURES 14 Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Veronica Astro and Antonio Adamo PART IV INFORMATIC TOOLS FOR STUDYING GERM CELLS DEVELOPMENT AND RECOMBINATION 15 Data Analysis Pipeline for scRNA-seq Experiments to Study Early Oogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Wei Ge, Teng Zhang, Yang Zhou, and Wei Shen 16 Mapping Meiotic DNA Breaks: Two Fully-Automated Pipelines to Analyze Single-Strand DNA Sequencing Data, hotSSDS and hotSSDS-extra. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Pauline Auffret, Bernard de Massy, and Julie A. J. Clément 17 “MeiQuant”: An Integrated Tool for Analyzing Meiotic Prophase I Spread Images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Julien Cau, Laurine Dal Toe, Akbar Zainu, Frédéric Baudat, and Thomas Robert Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
- 14. Contributors ANTONIO ADAMO • Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia VERONICA ASTRO • Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia PAULINE AUFFRET • Ifremer, IRSI, SeBiMER Service de Bioinformatique de l’Ifremer, Plouzané, France M. AZIM SURANI • Wellcome Trust/Cancer Research U.K. Gurdon Institute, Henry Wellcome Building of Cancer and Developmental Biology, Cambridge, UK; Physiology, Development and Neuroscience Department, University of Cambridge, Cambridge, UK YONI BAERT • Biology of the Testis Lab (BITE) Research Group, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium; In Vitro Toxicology and Dermato-Cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Brussels, Belgium FRÉDÉRIC BAUDAT • Institut de Génétique Humaine, University of Montpellier, CNRS, Montpellier, France MERIAM BOUBAKRI • Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands JULIEN CAU • Biocampus Montpellier, University of Montpellier, CNRS, INSERM, Montpellier, France YOLANDA W. CHANG • Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands LUIZA CHOJNACKA-PUCHTA • Department of Chemical, Biological and Aerosol Hazards, Central Institute for Labour Protection-National Research Institute, Warsaw, Poland SUSANA M. CHUVA DE SOUSA LOPES • Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands; Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium JULIE A. J. CLÉMENT • Institut de Génétique Humaine (IGH), Centre National de la Recherche Scientifique, UnivMontpellier, Montpellier, France; IHPE, Univ Montpellier, CNRS, IFREMER, Univ Perpignan Via Domitia, Perpignan, France MASSIMO DE FELICI • Department of Biomedicine and Prevention, Section of Histology and Embryology, Faculty of Medicine and Surgery, “Tor Vergata” University of Rome, Rome, Italy BERNARD DE MASSY • Institut de Génétique Humaine (IGH), Centre National de la Recherche Scientifique, UnivMontpellier, Montpellier, France NICHOLAS A. DEEBEL • Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA PRITHA DEY • Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy LUDOVICA DONADINI • Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy FEDERICA FRANCIOSI • Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy ix
- 15. x Contributors GUILLERMO GALDON • Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA WEI GE • College of Life Sciences, Qingdao Agricultural University, Qingdao, China RAFFAELE GEREMIA • Department of Biomedicine and Prevention, Section of Human Anatomy, University of Rome Tor Vergata, Rome, Italy MARK E. GILL • Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; Reproductive Medicine and Gynecological Endocrinology, University Hospital Basel, Basel, Switzerland ELLEN GOOSSENS • Biology of the Testis Lab (BITE) Research Group, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium JANMEJAY HINGU • Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA MASUMI HIRABAYASHI • Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi, Japan; The Graduate University of Advanced Studies, Aichi, Japan NAOKO IRIE • Wellcome Trust/Cancer Research U.K. Gurdon Institute, Henry Wellcome Building of Cancer and Developmental Biology, Cambridge, UK; Metabolic Systems Laboratory, Live Imaging Center, Central Institute for Experimental Animals, Kawasaki- ku, Kanagawa, Japan TOSHIHIRO KOBAYASHI • Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan; Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, Japan HUBERTUS KOHLER • Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland VALENTINA LODDE • Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy MARIA LÓPEZ PANADÉS • Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain; Department of Cell Biology, Physiology, and Immunology, Cytology and Histology Unit, Universitat Autònoma de Barcelona, Barcelona, Spain ALBERTO MARIA LUCIANO • Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy NOEMI MONFERINI • Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, University of Milan, Milan, Italy CHIARA NARO • Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, Rome, Italy; GSTeP-Organoids Research Core Facility, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy NIKOLETA NIKOU • Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain; Department of Cell Biology, Physiology, and Immunology, Cytology and Histology Unit, Universitat Autònoma de Barcelona, Barcelona, Spain MARIELLA OBERMEIER • Biology of the Testis Lab (BITE) Research Group, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium
- 16. Contributors xi MAMI OIKAWA • Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Laboratory of Regenerative Medicine, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan AREND W. OVEREEM • Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands ANTOINE H. F. M. PETERS • Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; Faculty of Science, University of Basel, Basel, Switzerland GUILLAUME RICHER • Biology of the Testis Lab (BITE) Research Group, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium THOMAS ROBERT • CBS (Centre de Biologie Structurale), Univ Montpellier, CNRS, INSERM, Montpellier, France CELINE M. ROELSE • Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands VERA ROGIERS • In Vitro Toxicology and Dermato-Cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Brussels, Belgium IGNASI ROIG • Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain; Department of Cell Biology, Physiology, and Immunology, Cytology and Histology Unit, Universitat Autònoma de Barcelona, Barcelona, Spain HOOMAN SADRI-ARDEKANI • Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA DOROTA SAWICKA • Department of Chemical, Biological and Aerosol Hazards, Central Institute for Labour Protection-National Research Institute, Warsaw, Poland CLAUDIO SETTE • Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, Rome, Italy; GSTeP-Organoids Research Core Facility, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy WEI SHEN • College of Life Sciences, Qingdao Agricultural University, Qingdao, China LAURINE DAL TOE • CBS (Centre de Biologie Structurale), Univ Montpellier, CNRS, INSERM, Montpellier, France TAMARA VANHAECKE • In Vitro Toxicology and Dermato-Cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Brussels, Belgium NING WANG • Department of Cell Biology and Physiology, University of Kansas Medical Center, Kansas City, KS, USA; Center for Reproductive Sciences, Institute for Reproductive and Developmental Sciences (IRDS), University of Kansas Medical Center, Kansas City, KS, USA AKBAR ZAINU • Institut de Génétique Humaine, University of Montpellier, CNRS, Montpellier, France TENG ZHANG • College of Life Sciences, Inner Mongolia University, Hohhot, China XIAOYU ZHANG • Department of Cell Biology and Physiology, University of Kansas Medical Center, Kansas City, KS, USA; Center for Reproductive Sciences, Institute for Reproductive and Developmental Sciences (IRDS), University of Kansas Medical Center, Kansas City, KS, USA YANG ZHOU • College of Life Sciences, Inner Mongolia University, Hohhot, China
- 17. Part I Isolation and Purification of Primordial (and) Germ Cells
- 18. Chapter 1 Isolation and Purification of Viable PGCs from Mouse Embryos Massimo De Felici Abstract In all organisms with sexual reproduction, sperm and oocytes derive from embryonic precursors termed primordial germ cells (PGCs) which pass on genetic information to subsequent generations. Studies aimed to unravel PGC development at molecular level in mammals can be traced at the early 1980s and were hampered by the difficulty in obtaining both sufficient quantities and purity of PGCs. For many labora- tories, the isolation and purification methods of PGCs at different stages from embryos are the most shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. In the present chapter, I focus on immunomagnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to 12.5 dpc embryos before their differentiation in oogonia/oocytes in female and prospermatogonia in male. Key words Primordial germ cells, MiniMACS, SSEA-1, CD-15, Gonadal ridges, AGM, FACS, OCT4, eGFP 1 Introduction In the mouse embryo, it is now well established that PGC precur- sors are specified from a small group of proximal epiblast cells and become identifiable as alkaline phosphatase (AP)- and PRDM1- positive cells at the base of allantois in the extraembryonic meso- derm at around 7.5 days post coitum (dpc) (for a review, see Ref. [1]). It is not clear if at this time these cells are part of a heteroge- neous population of pluripotent stem cells destinated to give rise to various lineage (for a review, see Ref. [2]). In any case, during gastrulation, PGCs can be traced within the developing hindgut endoderm at 7.75 dpc and into the aorta-gonad-mesonephros (AGM) region at 9.5–10.5 dpc. Between 11.5 and 12.5 dpc, PGCs become germline determined and initiate their sexual differ- entiation into prospermatogonia (male) and oogonia (female), instructed by the surrounding microenvironment of the gonadal Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology, vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 3
- 19. niche. From about 13.5 dpc, prospermatogonia undergo a tempo- rary GO mitotic arrest while oogonia enter the prophase of meiosis I as primary oocytes. 4 Massimo De Felici PGCs representing a key cell type for analysis of germ-soma differentiation as well as pluripotency at the molecular level. More- over, in the last years, the possibility to generate primordial germ cells–like cells (PGCLCs) in vitro able to generate male and female gametes from pluripotent stem cells of various mammalian species including humans (for a review, see Ref. [3]) increased exponen- tially the interest about such unique cell type. Today single-cell genome and transcriptome sequencing methods enabling the simultaneous molecular analysis of hundreds, or thousands of cells, inside heterogeneous populations are considerably increasing our capability to analyze the PGC genome and transcriptome (for example, see Refs. [4–6]. Nevertheless, for many laboratories, the isolation and purification methods of PGCs at different stages from embryos remain the most shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. Historically, the isolation and purification of PGCs from mouse embryos between 8.5 and 12.5 dpc were attempted using cellular, molecular, and transgenic approaches in parallel with the progress of technologies as summarized in Table 1. At these stages, PGCs are characterized by motility capability and active proliferation; they are for the most part single or form small clusters and do not form firm contacts with the surrounding extracellular matrix molecules and surrounding somatic cells. Both female and male mouse PGCs express surface markers such as tissue-nonspecific alkaline phospha- tase (TNAP), the carbohydrate epitopes bearing terminal Lewis X determinants (Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1, E-cad- herin, and the c-Kit and CXCR4 receptors. In principle, antibodies against all these surface molecules can be used to purify PGCs at these stages. Moreover, PGCs express several genes typical of plu- ripotent stem cells such as Pou5f1 (Oct4), Sox2, Nanog and of the germline such Nanos3, Dazl and Ddx4 (Vasa). Therefore, PGCs can be purified also from embryos of transgenic mice carrying reporter genes under the control of such genes (for example, see Refs. [12, 13]). In the present chapter, I focus on immunomagnetic cell sorting (iMACS) and fluorescence-activated cell sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to 12.5 dpc embryos using anti-SSEA-1 (CD15) MicroBeads and the transgenic mouse line Oct4ΔPE: eGFP (OG2), respectively.
- 20. Purification of Mouse PGCs 5 Table 1 Timetable of the methods used for the first time to isolate and purify mouse PGCs Heath J. K. (1978) [7] (Cell 15, 299–306) Manual dissection of gonadal ridges De Felici M. and McLaren A. (1982) [8] Percoll gradient McCarrey J. R. et al. (1987) [9] Immunopurification by FACS Massimo De Felici and Maurizio Pesce (1995) [10] Immunoaffinity adhesion Pesce, M., and De Felici, M. (1995) [11] Immunopurification by MACS Abe K. et al. (1996) [12] (Dev. Biol. 189, 468–472) Transgenic TNAPβ-geo PGCs by FACS-gal Szabò P. E. et al. (2002) [13] Transgenic Oct4ΔPE:eGFP PGCs by FACS Mayanagi T. et al. (2003) [14] Nicodenz gradient 2 Materials and Instruments 2.1 Isolation of PGCs by MiniMACS 1. 10.5–12.5 dpc pregnant females of desired genetic background (see Note 1). 2. 1xPBS, sterile, for dissecting. 3. Hanks’ Balanced Salt Solution (HBSS) or M2 (Sigma-Aldrich). 4. Trypsin/EDTA solution (0.025% trypsin and 0.01% EDTA) in PBS (Gibco). 5. DNase (1 mg/mL) solution (STEMCELL Technology). 6. Culture-tested fetal calf serum (FCS). 7. Poly-L-lysine-coated glass coverslips: soak coverslips in 200 μg/mL poly-L-lysine in distilled water for at least 1 h at r. t., wash three times in distilled water and air-dry. 8. Chromogenic solution for alkaline phosphatase (AP) detection: immediately before use dissolve 1 mg/mL fast red TR salt or fast blue BB salt (Sigma-Aldrich) in distilled water and add 40 μL/mL naphthol AS-BI or AS-MX alkaline solution (Sigma-Aldrich). 9. Fine scissors, dissection forceps and No. 5 watchmaker’s for- ceps, sterile. 10. Eppendorf or equivalent single channel 10–100 μL pipette. 11. 18- and 23-gauge needle and 1 mL syringe for tissues dissection. 12. 5 mL test tubes and 1.5 and 2 mL Eppendorf tubes. 13. Stereomicroscope (the author uses a Wild M5a from Leica, maximum magnification 100×, equipped with both incident and transmitted light sources, the latter preferably with a mov- able mirror).
- 21. 6 Massimo De Felici 14. Anti-SSEA-1 (CD15) MicroBeads (130-094-530, Miltenyi Biotech). 15. MACS buffer: phosphate-buffered saline (DPBS) without Ca2 + and Mg2 +, with 0.5% bovine serum albumin (BSA), and 2 mM EDTA (keep buffer cold at 2-8 °C and degas before use, as air bubbles could block the column). 16. MiniMACS™ Separator (Miltenyi Biotech). 17. MACS MultiStand (Miltenyi Biotech). 18. MACS MS separation columns (Miltenyi Biotech). 19. Class II Biological Hazard laminar flow hood (see Note 2). 2.2 Isolation of PGCs from OG2 Mice by FACS Materials listed in the previous section from 2 to 11 are required also in this procedure. 1. 10.5–12.5 dpc pregnant CD1 females previously mated with OG2 homozygous males (see Note 3). 2. Hanks’ Balanced Salt Solution (HBSS) or M2 (Sigma-Aldrich) plus 1 mg/mL BSA. 3. 5 mL FACS tubes compatible with flow cytometer. 4. Sorting flow cytometer (see Note 4). 3 Methods 3.1 Dissection of AGM and GRs At 10.5 dpc, the region dissected away from the embryo (hindgut, dorsal mesentery, and gonadal ridges) corresponds to the region termed AGM (aorta, gonad, mesonephros) containing the most part of migratory PGCs (about 1000/embryo, while at 11.5 and 12.5 dpc PGCs are obtained from the gonadal ridges (GRs, about 3000/and 8000/embryo) ([15], and our unpublished data). Embryonic sex can be determined only by PCR of genomic DNA using specific primers for 10.5 and 11.5 dpc samples (see Note 5) or by assessing gonad morphology for 12.5 dpc (Fig. 1). The procedure described in the following steps is routinely used in the author’s laboratory, but it can be varied according to the operator’s preference and experience. Steps 1. Lay the freshly killed animal on its back and soak the abdomen with 70% alcohol. CD1 mice, usually employed in the author’s laboratory, were housed and mated under conventional condi- tions (see Note 1). Positive plug test is considered 0.5-day post coitum. The developmental stage of embryos can be precisely
- 22. Purification of Mouse PGCs 7 Fig. 1 (a) Morphology of limb buds of the mouse embryo between 10.5 and 12.5 dpc. (b) Sex indifferent 11.5 dpc gonadal ridges (upper), 12.5 dpc testis (right), 12.5 dpc ovary (left) determined by observing the morphology of the hind limb bud (Fig. 1). 2. Pinch up the skin of the abdomen region between thumb and forefinger of both hands and pull it toward the head and tail until the abdomen is completely exposed and the fur is well out of the way. 3. Using forceps and fine scissors, cut the body wall, push the coil of gut out of the way, and locate the two horns of the uterus. 4. Remove the uterus intact by cutting across the cervix and the two uterotubal junctions and trim away the fat and mesentery with fine scissors. 5. Transfer the uterus in a petri dish in a volume of PBS sufficient to completely immerse the tissues and remove any residual fat and mesentery. 6. Transfer the uterus to a second dish of fresh medium and dissect the embryos with attached placenta by cutting the anti- mesometrial wall of the uterus with the tips of scissors. Remove the embryo’s membranes by cutting at the junction of the placenta with watchmaker’s forceps. 7. Cut with scissors the anterior half of the embryo just below the armpits and transfer the posterior half to a dish of fresh medium, 8. Under a stereomicroscope, turn back such posterior half and hold it with No. 5 watchmaker’s forceps while making a cut along the ventral midline. 9. For 10.5 dpc embryos, using the tips of No. 5 watchmaker’s forceps, remove viscera paying attention does not remove the
- 23. 8 Massimo De Felici dorsal mesentery and the hindgut. Peel away these and asso- ciated GRs (gonad + mesonephros) from the dorsal region of the embryo and transfer them into HBSS or M2 (Fig. 2). 10. For 11.5 and 12.5 dpc embryos, scoop out liver and any remnants of intestine with No. 5 watchmaker’s forceps and identify the GRs lying on the dorsal wall of the embryos on either side of the abdominal aorta. Slide the tips of the No. 5 watchmaker’s forceps behind each GR and remove them from the embryo. 11. Transfer the GRs to HBSS or M2 and separate the gonads from mesonephroi using 25G needles. 3.2 Isolation of 10.5– 12.5 dpc PGCs by MiniMACS In this method, PGCs are separated from the other cell types by positive selection. This means that they are labeled with superpar- amagnetic microbeads conjugated to highly specific antibodies. During separation, the magnetically labeled PGCs are retained within a column, while unlabeled cells flow through. After a wash- ing step, the column is removed from the magnetic field of the separator, and PGCs are eluted. Positive selection can be performed by direct or indirect magnetic labeling. The following procedure describes a direct labelling of PGCs by anti-SSEA-1 MicroBeads purchased from Miltenyi Biotechnology. The simplicity and low cost make the MiniMACS separation procedure the method of choice for rapidly obtaining reasonable numbers of purified germ cells and somatic cells that can be used immediately for biochemical studies or be grown in suitable in vitro culture systems. In 2–3 h of work, one person should be able to isolate from 10 embryos of 10.5, 11.5, and 12.5 dpc (a reasonable yield from a pregnant CD1 female) about 5000–7000 (purity 70–80%), 10,000–15,000, and 40,000–50,000 PGCs (purity 85–95%), respectively (Fig. 3). The presence of supermagnetic microbeads coated with antibodies on the cell surface might be harmful for certain cell functions. However, we cultured PGC purified by MiniMACS for several days without any apparent dele- terious effect (for example see Ref. [16]). Steps 1. 10.5 dpc AGM (usually 10–20) or 11.5–12.5 gonads (usually 20–40) are transferred with 100 μL Eppendorf pipette in mini- mal amount of medium into a 1.5 mL Eppendorf tube contain- ing 0.5 mL of trypsin-EDTA. 2. After about 5 min incubation at 37 °C carefully remove the solution and wash the tissues twice with 1 mL of HBSS or M2 + 5% FCS, allowing them to settle on the bottom of the tube. Leave the tissue in about 100 μL of medium and pipette up and down with the Eppendorf pipette for their complete
- 24. Purification of Mouse PGCs 9 Fig. 2 Dissected AGM (aorta, gonad, mesonephros) from 10.5 dpc embryo Fig. 3 Mouse 12.5 dpc PGCs stained with anti-SSEA-1 (CD15) FITC antibodies dispersion (about 10 strokes are usually sufficient). Ensure a single cell suspension by passing through an 18- and 23-gauge needle. 3. Add 1 mL of MACS buffer containing 10 μg/mL DNase, centrifuge at 500 ×g for 10 min at 4 °C. Remove supernatant and resuspend the pellet in 80 μL of buffer with the Eppendorf pipette (see Note 6). 4. Add 20 μL anti-SSEA-1 MicroBeads and incubate with contin- uous agitation for 45–60 min at 4 °C. 5. Dilute the cells to a final volume of 1.5 mL with MACS buffer and transfer on top of an AS column previously washed, filled with the buffer, and placed in the magnetic field of a Mini- MACS separation unit (Fig. 4). 6. Collect the effluent as SSEA-1 negative cells (somatic cells) and wash the column twice with 1 mL buffer into 5 mL collecting tube. 7. Remove the column from the magnetic field and, using the plugger supplied with the column, flush out with 1.5 mL buffer the cells retained by the column (SSEA-1 positive PGCs) in a 2 mL Eppendorf tube.
- 25. 10 Massimo De Felici Fig. 4 A MiniMACS separation unit form Miltenyi Biotec 8. To assess PGC purity, a small sample of the cell suspension (20–30 μL) can be transferred onto a polylysinated coverglass and stained for AP. 3.3 Isolation of 10.5– 12.5 dpc PGCs by FACS At these developmental stages, FACS can be used to purify viable PGCs labeled with antibodies anti-SSEA-1 (CD15) or other sur- face markers (e.g., EMA-1, E-cadherin, Kit) conjugated with vari- ous fluorochromes commercially available from several companies. Alternatively, PGCs can be isolated from transgenic mice expressing reporter genes under the control of early PGCs genes (e.g., Pou5f1, Prdm1, and Dppa3). Here we describe PGC purification from OG2 transgenic mice (Oct4ΔPE:eGFP) expressing enhanced green fluo- rescent protein (eGFP) under the control of the Pouf1 (Oct4), promoter and distal enhancer [17] (Fig. 5a). Steps 1. After steps 1 and 2 of Subheading 3.2 Isolation of PGCs by MiniMACS, add 1 mL of HBSS plus 1 mg/mL BSA contain- ing 10 μg/mL DNase, centrifuge at 500 ×g for 10 min at 4 °C. Remove supernatant, and resuspend the pellet in 0.5 mL of HBSS with the Eppendorf pipette (see Note 6). 2. Transfer the cell suspension into a FACS tube on ice and proceed to flow cytometer. 3. Cell debris and dead cells are excluded from the analysis based on scatter signals and propidium iodide fluorescence. Viable cells are gated based on forward scatter (FSC) vs. side scatter (SSC) profile. eGFP vs. FSC will delineate eGFP-positive PGC
- 26. Purification of Mouse PGCs 11 Fig. 5 (a) 12.5 dpc ovary and testis from OG2 mouse. Note a spotty pattern of fluorescence in the ovary, whereas a wavy pattern reflecting testicular cords formation is visible in the testis. (b) Representative FACS profile of disaggregated gonads isolated from 12.5 dpc CD1/OG2 embryos. Right: FSC-A/SSC-A dot plots of the cell suspension showing the cell population selected for fluorescence analysis. Left: GFP-A/FSC-A showing two eGFP positive PGC populations to be sorted displaying high or dull fluorescence; Inset: a group of PGCs positive for eGFP-OCT4 population of cells and the remaining eGFP negative cells as somatic cells (Fig. 5b), (see Note 7). 4. To assess PGC purity, a small sample of the cell suspension (20–30 μL) can be transferred onto a polylysinated coverglass and observed onto a fluorescence phase contrast microscope. The purity of sorted PGC population was usually 98%. 5. Sorted cells can be used immediately for biochemical studies or be grown in suitable in vitro culture systems.
- 27. ) 12 Massimo De Felici 4 Notes 1. Use the mouse strain of your choice, although we have fond that outbred strains such as MF1 and CD1 mice give larger numbers of PGCs than some inbred strains (e.g., BalbC). The procedure described here and the mean number of PGCs obtained are from CD1 embryos. Moreover, consider that PGC number can vary considerably among embryos. 2. The safety cabinet is necessary to carry out the whole procedure if sterility must be maintained. 3. The OG2 mice can be purchased from Jackson Laboratory, Cat # 003715. To ensure large litter sizes and large numbers of germ cells per gonad homozygous OG2 male can be mate with CD1 females. For the present protocol, usually, a minimum of two litters is pooled. 4. For the present procedure, cell sorting was performed on a FACSAria II (Becton Dickinson) interfaced with the FACS- DiVa 6.0 Software. 5. If sexing is required, each embryo is dissected into a 24-well plate containing warm DMEM+10% FCS and a tail tip is taken for PCR sexing. We used genomic PCR of mouse Ube1 [18] or Xlr and Sly [19] genes. Once results are available (2–3 h embryos are pooled based on sex. 6. For optimal performance, it is important to obtain a single-cell suspension. Check a small sample of the cell suspension under a microscope and if necessary, pass the suspension through a 23-gauge needle once more or filter through a 40 μm cell strainer (BD Falcon). The percent of PGCs in the cell suspen- sion estimated by APase staining (see step 8) was less than 0.1% and approximately 5% and 30–35% at 10.5, 11.5, and 12.5 dpc, respectively. 7. Although the most part of PGCs showed high eGFP, we and others observed a little population of weakly eGFP positive cells. At 10.5 and 11.5 dpc, these latter represented between 9% and 12% of the whole eGFP positive cells. Hypotheses about such minority PGC population are discussed in [20]. Although FACS is the method of choice for cell sorting when high cell purity is needed, it suffers, however, from (sometimes very) high losses, requires trained personnel, and is expensive to own and operate. The estimation of PGC recov- ery under the present conditions varied from 65% to 70%. This means that from 10 embryos of 10.5, 11.5, and 12.5 dpc heterozygotes CD1/OG2 females, about 5000, 15,000, and 35,000 PGCs can be obtained, respectively. Finally, PGCs
- 28. Purification of Mouse PGCs 13 isolated by FACS from transgenic fluorescent proteins should not have any potential adverse effect caused by antibodies bound to their cell surface. References 1. De Felici M (2016) The formation and migra- tion of primordial germ cells in mouse and man. Results Probl Cell Differ 58:23–46 2. Mikedis MM, Downs KM (2014) Mouse pri- mordial germ cells: a reappraisal. Int Rev Cell Mol Biol 14(309):1–57 3. Hayashi M, Kawaguchi T, Durcova-Hills G, Imai H (2017) Generation of germ cells from pluripotent stem cells in mammals. Reprod Med Biol 17:107–114 4. Guo F, Yan L, Guo H, Lin Li L et al (2015) The transcriptome and DNA methylome land- scapes of human primordial germ cell. Cell 161:1437–1452 5. Zhao ZH, Ma JY, Meng TG, Wang ZB et al (2020) Single-cell RNA sequencing reveals the landscape of early female germ cell develop- ment. FASEB J 34:12634–12645 6. Ge W, Wang JJ, Zhang RQ, Tan SJ et al (2021) Dissecting the initiation of female meiosis in the mouse at single-cell resolution. Cell Mol Life Sci 78:695–713 7. Heath JK (1978) Characterization of a xeno- geneic antiserum raised against the fetal germ cells of the mouse: cross-reactivity with embry- onal carcinoma cells. Cell 15:299–306 8. De Felici M, McLaren A (1982) Isolation of mouse primordial germ cells. Exp Cell Res 142:476–482 9. McCarrey JR, Hsu KC, Eddy EM, Klevecz RR, Bolen JL (1987) Isolation of viable mouse pri- mordial germ cells by antibody-directed flow sorting. J Exp Zool 242:107–111 10. De Felici M, Pesce M (1995) Immunoaffinity purification of migratory mouse primordial germ cells. Exp Cell Res 26:277–279 11. Pesce M, De Felici M (1995) Purification of mouse primordial germ cells by MiniMACS magnetic separation system. Dev Biol 170: 722–725 12. Abe K, Hashiyama M, Macgregor G, Yama- mura K (1996) Purification of primordial germ cells from TNAPbeta-geo mouse embryos using FACS-gal. Dev Biol 180:468– 472 13. Szabò PE, Hübner K, Schöler H, Mann JR (2002) Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 115:157–160 14. Mayanagi T, Kurosawa R, Ohnuma K, Ueyama et al (2003) Purification of mouse primordial germ cells by Nycodenz. Reproduction 125: 667–675 15. Tam P, Snow M (1981) Proliferation and migration of primordial germ cells during com- pensatory growth in mouse embryos. J Morph Exp Morph 64:133–147 16. Farini D, Scaldaferri ML, Iona S, La Sala G, De Felici M (2005) Growth factors sustain primor- dial germ cell survival, proliferation and enter- ing into meiosis in the absence of somatic cells. Dev Biol 285:49–56 17. Yoshimizu T, Sugiyama N, De Felice M, Yeom YI et al (1999) Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ 41: 675–684 18. Chuma S, Nakatsuji N (2001) Autonomous transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130- mediated signaling. Dev Biol 229:468–479 19. McFarlane L, Truong V, Palmer JS, Wilhelm D (2013) Novel PCR assay for determining the genetic sex of mice. Sex Dev 7:207–211 20. Scaldaferri ML, Klinger FG, Farini D, Di Carlo A et al (2015) Hematopoietic activity in puta- tive mouse primordial germ cell populations. Mech Dev 136:53–63
- 29. Chapter 2 Purification and Transfection Methods of Chicken Primordial Germ Cells Luiza Chojnacka-Puchta and Dorota Sawicka Abstract Primordial germ cells (PGCs) play a special role in the vertebrate life cycle since they are the precursors of germ cells through which the genome is passed to the next generations. PGCs are found in different locations and variable numbers in the chick embryo, as in other species, depending on the developmental stages. Here, we describe in detail a method based on the Percoll gradient, routinely used in our laboratory, allowing us to obtain from blood and gonad anlages significant numbers of viable PGCs which can be successfully cultured or efficiently genetically modified. Key words Chicken primordial germ cells, Percoll density gradient, Electroporation, Lipofection, Immuno-cyto-staining 1 Introduction Primordial germ cells (PGCs) play a special role in the vertebrate life cycle since they are the precursors of ova and spermatozoa through which the genome is passed to the next generations. In birds the germline is established early in the embryo, likely by maternal specification [1]. In the chick embryo, PGC precursors are initially detected as a scattered pattern in the area of pellucida, the central region of the Eyal-Giladi and Kochav stage X embryo [2]. From this position, these cells are translocated to the anterior extraembryonic region called the germinal crescent, where they, now termed PGCs, are incorporated into the forming extraembry- onic vascular network [stages 8–10 according to Hamburger and Hamilton (HH 8–10)] [3], and start to circulate within the blood- stream (stage HH 11). Reaching the gonadal anlage region, PGCs exit the blood vessels and colonize the gonads (stage HH 16). About 30 PGCs are found at X stage embryo in the area of pellu- cida, 200–250 in the germinal crescent, and more than 1000 are in the gonads after embryo incubation for 7 days (stages 26–29 HH) Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology, vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 15
- 30. [5]. Considering such dynamics localization, at the 10 HH stage, PGCs can be obtained from blastoderm, at 13–17 HH stages (2.5- to 3-day-old embryos) from blood and at stages 26–29 HH stages (5.5- to 6-day-old embryos) directly from the embryonic gonads. PGCs isolated from these stages have been successfully used for transfection [4, 5] or cryoconservation [6]. 16 Luiza Chojnacka-Puchta and Dorota Sawicka Purification methods for chick PGCs range from density gra- dients using Nycodenz [7], Ficoll [8], or Percoll [9] to immuno- magnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) [11]. Chick PGCs were purified from blood using ammonium chloride-potassium (ACK) lysis buffer to eliminate red blood cells, followed by in vitro culture [10]. Using such methods, the purity of PGCs overcame 90% at best. Chick PGCs can be identified based on morphological criteria, i.e., large size and presence of a large amount of glycogen in the cytoplasm [12]. Glycogen granules can be evidenced also by peri- odic acid-Schiff (PAS) staining [13, 14]. Moreover, chick PGCs were stained for alkaline phosphatase activity, a classical PGC marker also in mammalian species [15], and for many immunolog- ical epitopes for surface glycoproteins. The best known and char- acterized is SSEA-1 (Stage-specific Embryonic Antigen-1) [16], which is effective only after stage 10 HH. Other immunological markers are EMA-1 (Epithelial Membrane Antigen) [17] expressed from the crescent stage [18] and MVH (Mouse Vasa Homolog) [19]. Interestingly, all these immunological markers are common to mammalian PGCs (see Chap. 1, by M. De Felici, in the present book) Here we describe a simple and rapid method based on Percoll density gradient suitable to purify chicken PGC from blood and early embryonic gonads that can be successfully transfected using the electroporation/lipofection method for germline manipula- tion. Details are in Table 1. Table 1 Average numbers of chicken embryos, PGCs isolated from blood and gonads, purity and efficiency of transfection methods Type of PGCs Number of embryosa Number of total cells after isolation Number of total after Percoll purification Purity of PGCs (%) Electroporation efficiency (%) Lipofection efficiency (%) bPGCs 60 11.6 × 106 1.76 × 106 64 75 50 gPGCs 30 4.53 × 106 7.56 × 105 N/A 73 39 Data obtained in our experiments a The number of embryos used to isolate PGCs depends on the quality of embryos and operator skills. Some of them can be unfertilized or undeveloped
- 31. Purification and Transfection of PGCs 17 2 Materials 2.1 Reagents 1. Freshly fertilized eggs. 2. 0.2 M EDTA: Prepare 100 mL of 0.2 M EDTA by solving 7.44 g EDTA in 100 mL distillated water. Mix and adjust pH 7.4 with NaOH. This solution must be autoclaved. Prepare 10 mL of 0.1 mM EDTA by dilution of 5 μL 0.2 M EDTA in 10 mL 1× PBS (without calcium or magnesium) and mix. 3. 0.25% trypsin EDTA: Dilute 40 μL 0.25% trypsin EDTA solu- tion in 960 μL OptiMEM I to obtain 0.01% trypsin; make fresh every time. 4. Antibiotic: Penicillin/streptomycin (10,000 U/mL). 5. OptiMEM I supplemented with 5% FBS. For 50 mL: add 2.5 mL FBS to 47.5 mL of OptiMEM I. Store at 4 °C. 6. Percoll 100% to prepare density solutions: 50%, 25%, and 12.5%. Make the steps by diluting the 100% Percoll, as shown in Table 2. All solutions are freshly made; phenol red is used to provide visual contrast for different phases. 7. Culture complex medium: OptiMEM I, 2% CS—chicken serum, 10% FBS—fetal bovine, 20 ng/mL bFGF—basic fibro- blast growth factor, 9 ng/mL mLIF—murine leukemia inhibi- tory factor, 5 ng/mL hSCF—human stem cell factor, 1% antibiotic (and selective antibiotic). 2.2 Equipment 1. Egg incubator. 2. Cell tram air/oil pump with capillary holder and fine glass micropipette (90 × 135 mM). 3. Dissection microscope (a binocular stereoscopic dissection microscope is preferred). 4. Centrifugator with swinging-bucket rotor and buckets. 5. CO2 incubator (5%). 6. Cell counter/hemocytometer. Table 2 Steps to prepare Percoll solutions Step Percoll final concentration (%) OptiMEM I with 1% FBS (mL) 100% Percoll (mL) 1. 50 10 10 2. 25 15 5 3. 12.5 17.5 2.5 The amounts of medium OptiMEM I with 5% FBS and Percoll are calculated for a final volume of 20 mL
- 32. 18 Luiza Chojnacka-Puchta and Dorota Sawicka 7. 15 mL tubes, 1.5 mL Eppendorf tubes. 8. Inverted/fluorescent microscope. 9. 4-well plates, petri dishes. 10. Electroporator, 0.4 mL cuvettes. 3 Methods Prepare a workplace, where cells will be isolated from chicken embryos by wiping the table with a disinfectant or 70% ethanol solution. Also wipe the elements of the stereoscope and cell tram air/oil pump. Perform all procedures using gloves and wear a lab coat. 3.1 Preparation Embryos to Isolate of PGCs 1. Incubation eggs. Incubate freshly fertilized laid chicken eggs (egg or meat breed) at 37.8 °C and 60–62% relative humidity for 50–56 h to achieve chicken embryos at 14–16 HH and for 6 days to obtain chicken embryos at 28–29 HH (see Note 1). 3.2 Isolation of PGCs from Bloodstream (bPGCs) 1. Prepare 0.5 mL of 0.1 mM EDTA solution in 1 × PBS and supplement with 1% antibiotic. Prepare the cell tram air/oil pump and put the fine glass micropipette into a capillary holder. Fill the tip of the fine glass micropipette with the prepared solution. EDTA solution will prevent the drawn blood from clotting. 2. Take out chicken eggs from the incubator and wipe with a 70% solution of ethanol (see Note 2). 3. Gently transfer the contents of the egg to a sterile petri dish. Be careful not to damage the embryo. Transfer the petri dish under the stereoscope. 4. Take blood from the dorsal aorta of embryos (HH stages 14–16) using fine glass micropipette and suspend into EDTA solution to obtain suspension-contained bPGCs and blood counts (Fig. 1). Afterward, centrifuge cell suspension (400 × g, 5 min) and resuspend in medium (0.5 mL) Opti- MEM I supplemented with 5% FBS and 1% antibiotic. 5. Stain cells with 0.4% trypan blue (1:1) and count in a hemocy- tometer under an inverted microscope. 3.3 Isolation of PGCs from Gonads (gPGCs) 1. Prepare 1 × PBS containing 1% antibiotic at room temperature (RT). 2. Take chicken eggs from incubator and wipe with a 70% ethanol solution.
- 33. Purification and Transfection of PGCs 19 Fig. 1 Suspension of cells isolated from bloodstream of chicken embryos (HH stages 14–16) containing bPGCs and blood counts (20×) Fig. 2 Steps/scheme of isolation suspension of cells including gPGCs from gonads of 6-day-old chicken embryos. (a) chicken embryo on the 6-th day of incubation, (b) mesonephros with gonads (6.3×), (c) isolated gonads (40×), (d) suspension of cells containing gPGCs (20×) 3. Transfer the embryo (28–29 HH) (Fig. 2a) to a petri dish and purify the remains of the yolk by washing several times with 1 × PBS containing 1% antibiotic. 4. Transfer the embryo to a clean petri dish and isolate mesoneph- ros under a stereoscope (Fig. 2b). 5. Cut along the inner edge of the mesonephros with a 0.4 mM blade to isolate gonads (Fig. 2c) and transfer gonads into the tube with 1 × PBS containing 1% antibiotic.
- 34. 20 Luiza Chojnacka-Puchta and Dorota Sawicka 6. Mash the gonads with an insulin needle to increase the surface available for the enzyme. 7. Pipette vigorously and transfer all content to the new tube. 8. Centrifuge (400 × g, 5 min). 9. Add 1 mL of 0.01% trypsin and digest for 1 min at 37 °C in a heating block placed in a laminar airflow chamber. Inactivate the enzyme by adding 100 μL of FBS. 10. Centrifuge suspension of cells, including gPGCs (400 × g, 5 min), and resuspend in 0.5 mL medium OptiMEM I supple- mented with 5% FBS and 1% antibiotic. The obtained cells are presented in Fig. 2d. 11. Stain cells with 0.4% trypan blue (1:1) and count in a hemocy- tometer under an inverted microscope. 3.4 Purification and Identification of PGCs 1. Prepare Percoll solutions: 50%, 25%, and 12.5% in medium OptiMEM I supplemented with 5% FBS. Form density gradi- ent by layering 3 mL each of 50%, 25%, and 12.5% density of Percoll in a clear 15 mL conical centrifuge tube (see Note 3). 3.4.1 Percoll Purification 2. Suspend cells isolated from the bloodstream or gonads in 3 mL of OptiMEM I with 5% FBS. 3. Place suspension of cells (3 mL) on the top of the density gradient (Fig. 3) and centrifuge at 1.160 g for 20 min at 20 ° C (see Note 4). 4. Gently collect bPGCs or gPGCs between 50% and 25% Percoll layers (see Note 5). 5. Wash PGCs twice in medium OptiMEM I (with 5% FBS, 1% antibiotic). 6. Resuspend the cell pellet in medium OptiMEM I (with 5% FBS, 1% antibiotic), and centrifuge at 400 × g for 5 min at RT. 7. Gently remove supernatant, cells are ready for further proces- sing or other analysis. The purified PGCs isolated from the bloodstream or gonads are presented in Fig. 4. 3.4.2 Identification of PGCs 1. Fix purified 10 μL suspension PGCs in 10 μL 4% glutaralde- hyde for 5 min to dry. PAS Staining 2. Rinse twice in 1 × PBS. 3. Immerse cells in a 0.5% aqueous periodic acid solution for 5 min at room temperature. 4. Wash samples twice with 1 × PBS and incubate with Schiff’s Reagent for 15 min at RT. 5. Wash samples twice with 1 × PBS and observed under a light microscope (Fig. 5).
- 35. Purification and Transfection of PGCs 21 Fig. 3 Percoll gradient with a layer of cells isolated from the bloodstream of embryos at 14–16 HH contained suspension of bPGCs and blood counts (arrow). The bottom layer (50% Percoll solution) was stained by phenol red Immunocytochemistry 1. Take 100 μL purified cells (approx. 4.5 × 105 cells) and add 1 mL 1 × PBS, centrifuge at 400 × g for 5 min, and resuspend in 1 × PBS to rinse culture medium. 2. The PGCs suspension fix in 1% paraformaldehyde for 15 min. 3. Wash in 1 × PBS and centrifuge at 400 × g for 5 min. 4. Block cells with 2.5% goat serum for 30 min at RT. 5. Centrifuge at 400 × g for 5 min.
- 36. 22 Luiza Chojnacka-Puchta and Dorota Sawicka Fig. 4 The PGCs isolated from chicken embryos and purified via Percoll centrifugation density. PGCs are round cells ranging in diameter from 10 and 14 μM with a large nucleus and large, clearly visible vacuoles. (a) bPGCs (20×) and (b) gPGCs (40×) Fig. 5 The purified chicken PGCs stained with PAS. Positive results of staining (red- stained cells) indicate the presence of glycogen vesicles in the PGCs, which confirms their origin. (a) bPGCs (20×) and (b) gPGCs (40×) 6. Incubate cells with an antibody against a specific PGC marker (anti-stage-specific embryonic antigen 1 (SSEA-1)-FITC/ PerCP) for 1 h according to the manufacturer’s protocol (see Note 6). 7. Observe samples under fluorescent microscope at an excitation wavelength of 488 nM using a 515/30 filter or 682/33 (respectively for used fluorochromes) (Fig. 6). 3.5 Transfection Methods and Culture of PGCs 1. Centrifuge purified PGCs at 300 × g for 3 min and resuspend in medium without any additions (0.45 mL). 2. Add plasmid (20 μg) and transfer cells to 0.4 mM cuvette. 3.5.1 Electroporation 3. Perform electroporation under conditions: 200 V, 900 μF. 4. Transfer cells into the well of a 4-well plate and culture for 24 h (37 °C and 5%, CO2). 5. After 24 h, replace the medium with a culture complex medium (see Notes 7 and 8).
- 37. Purification and Transfection of PGCs 23 Fig. 6 The purified chicken PGCs subjected to immunofluorescence staining with antibodies for the specific cell surface antigen SSEA-1 after excitation wavelength of 488 nM. Staining of PGCs in green by anti-SSEA1- FITC (a) and red by anti-SSEA1-PerCp (b) confirmed their identity (20×) 3.5.2 Lipofection 1. Seed isolated and purified PGCs into 4-well plate. 2. All reagents: X-tremeGENE HP DNA Transfection Reagent, DNA and medium must be at RT (15–25 °C) (see Note 9). 3. Dilute DNA with medium OptiMEM I without any additions to a final concentration of 1 μg plasmid DNA/100 μL medium. 4. Transfer 100 μL of medium containing 1 μg DNA into a sterile tube. 5. Briefly vortex vial of Transfection Reagent and add 3 μL directly into the medium containing DNA from step 3. Mix gently. 6. Incubate for 15 min at RT. 7. Add transfection complex to PGCs in a dropwise manner. Gently shake the wells. 8. Incubate cells for 24 h. 9. Change medium to culture complex medium (see Notes 7 and 8). 4 Notes 1. Prepare the incubator with conditions: 37.8 °C and 60–62% relative humidity. At least 50 eggs for one isolation is needed. The most important thing is the temperature in the egg incu- bator. Plug in and turn on the incubator. Wait for the tempera- ture to stabilize. Remember, the more eggs you have, the longer it will take to get to the temperature and stabilize. Because the warm air naturally rises to equalize the warm air in each part of the incubator, install a fan with air circulation
- 38. 24 Luiza Chojnacka-Puchta and Dorota Sawicka best. Also, we recommend using a hygrometer, a device that measures humidity in the air. To increase the humidity, put a container with distilled water into the incubator. 2. Place the eggs in a horizontal position and leave for 5–10 min, allowing the embryo to assume the appropriate position when it is released from the egg. 3. 100% Percoll stored at 4 °C should have pH 8.5–9.5. We find that Percoll solutions is best to prepare fresh each time, before density gradient centrifugation. They need to be used at room temperature. Use phenol red to provide visual contrast for different phases. Add drop-by-drop directly to each 3 mL of load density successively: 50%, 25% and 12.5% into clear 15 mL tube, avoiding bubble formation. 4. Carefully layer cells onto the top of the 12.5% layer immediately before centrifugation. Place the gradient into a swinging bucket rotor carefully and centrifuge. 5. Carefully remove the gradient and collect PGCs from above the red layer between 50 and 25 phases. To make collection easier, first, remove the top layer. 6. We used an antibody sc-21702 Santa Cruz Biotechnology, Santa Cruz, CA, USA. 7. Change medium every 3 days and culture cells prior to further use (injection or cryoconservation). We recommend using OptiMEM I medium with supplements: 2% CS, 10% FBS, 20 ng/mL bFGF, 9 ng/mL mLIF, 5 ng/mL hSCF, 1% antibi- otic and selective antibiotic. 8. Check the effectiveness of electroporation PGCs based on viability if you use selective antibiotics or other markers. Add selective antibiotic after transfection to obtain stable transfec- tants. We used genetycin (G418) at a concentration of 50 μg/mL. 9. Perform lipofection of PGCs with the use of one of the com- mercially available lipofectants. In our laboratory we use the X-tremeGENE HP DNA Transfection Reagent (Roche) and lipofection protocol according to the manufacturer’s instruc- tions. We recommend the use of a 3:1 rate. This procedure does not require the removal of lipofectant (e.g., X-tremeGENE HP DNA Transfection Reagent or FUGENE 6 Transfection Reagent), which may be needed for the other reagents. Acknowledgments This paper was partially supported by resources from the Ministry of Education and Science and also funds intended for the
- 39. maintenance and progress of research with didactic potential. Task no. I-61. This work was also partially supported by the National Science Center (NCN 2018/02/X/NZ9/00997 2018). Purification and Transfection of PGCs 25 References 1. van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Brashaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ (2006) Germline transmission of genetically modified primordial cells. Nature 441:766–769 2. Eyal-Giladi H, Kochav S (1976) From cleavage to primitive streak formation: a complementary normal table and new look at the first stages of the development of the chick. I. General mor- phology. Dev Biol 49:321–337 3. Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92 4. Naito M, Sano A, Harumi T, Matsubara Y, Kuwana T (2004) Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blasto- derms and detection of germline chimaerism by PCR. Br Poult Sci 45:762–768 5. Chojnacka-Puchta L, Sawicka D, Lakota P, Plucienniczak G, Bednarczyk M, Plucienniczak A (2015) Obtaining chicken primordial germ cells used for gene transfer: in vitro and in vivo results. J Appl Genet 56(4):493–504 6. Sawicka D, Chojnacka-Puchta L, Zielinski M, Plucienniczak G, Plucienniczak A, Bednarczyk M (2015) Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells. Cell Mol Biol Lett 20(1):143–159 7. Zhao DF, Kuwana T (2003) Purification of avian circulating primordial germ cells by nyco- denz density gradient centrifugation. Br Poult Sci 44:30–35 8. Park TS, Jeong DK, Kim JN, Song GH, Hong YH, Lim JM, Han JY (2003) Improved germ- line transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod 68: 1657–1662 9. Oishi I (2010) Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation. Biosci Biotechnol Biochem 74(12): 2426–2430 10. Yamamoto Y, Usui F, Nakamura Y, Ito Y, Tagami T, Nirasawa K, Matsubara Y, Ono T, Kagami H (2007) A novel method to isolate primordial germ cells and its use for the gener- ation of germline chimeras in chicken. Biol Reprod 77(1):115–119 11. Mozdziak PE, Angerman-Stewart J, Rushton B, Pardue SL, Petitte JN (2005) Iso- lation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult Sci 84:594–600 12. Ginsburg M, Eyal-Giladi H (1986) Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. J Embryol Exp Morphol 95:53–71 13. Meyer DB (1960) Aplication of the periodic acid–Schiff technique to whole chick embryos. Biotech Histochem 35:83–89 14. Macdonald J, Glover JD, Taylor L, Sang HM, McGrew MJ (2010) Characterisation and germline transmission of cultured avian pri- mordial germ cells. PLoS One 5:e15518 15. Quaye Mensah B (2023) Ligamentum arterio- sum and its telocytes: an ultrastructure descrip- tion. Anat Rec (Hoboken) 306(1):187–192. https:/ /doi.org/10.1002/ar.25052. Epub 2022 Aug 6. PMID: 35904054 16. Resnick JL, Bixler LS, Cheng L, Donovan PJ (1992) Long-term proliferation of mouse pri- mordial germ cells in culture. Nature 359:550– 551 17. Urven LE, Erickson CA, Abbott UK, McCar- rey JR (1988) Analysis of germ line develop- ment in the chick embryo using an anti-mouse EC cell antibody. Development 103:299–304 18. Karagenc L, Cinnamon Y, Ginsburg M, Petitte JN (1996) Origin of primordial germ cells in the prestreak chick embryo. Dev Genet 19: 290–301 19. Tsunekawa N, Naito M, Sakai Y, Nishida T, Noce T (2000) Isolation of chicken vasa homolog gene and tracing the origin of pri- mordial germ cells. Development 127:2741– 2750
- 40. Chapter 3 Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells (SSCs) Janmejay Hingu, Guillermo Galdon, Nicholas A. Deebel, and Hooman Sadri-Ardekani Abstract Preservation of human spermatogonial stem cells (SSCs) may be suitable for young male patients at risk of male infertility due to various causes, such as gonadotoxic treatment or genetic diseases. With optimal cryopreservation, cell viability can be retained to reestablish spermatogenesis in the future through autolo- gous transplantation or in vitro differentiation of SSCs. This protocol outlines techniques to optimize the SSCs isolation and in vitro culture. With particular emphasis on the microscopic characteristics encoun- tered, this protocol outlines a broader approach to processing tissues with varying morphologies among patients. Key words Male infertility, Spermatogonial stem cell (SSC), Isolation, Propagation 1 Introduction Infertility is the inability to conceive after 12 months of unpro- tected intercourse and remains a public health issue for both men and women with equal prevalence. While many etiologies contrib- ute to an infertility diagnosis, the male factor is present in approxi- mately 50% of infertile couples and is the sole causative factor in 20–30% of couples [1]. Azoospermia (absence of sperm on two consecutive centrifuged semen analyses) is the most severe form of male infertility, ultimately leaving couples with limited options for biological paternity in the setting of a negative microdissection testicular sperm extraction procedure (mTESE) [2]. Despite a negative sperm retrieval, testicular biopsies showing hyposperma- togenesis, maturation arrest, or Sertoli cell only (SCO) may still present putative spermatogonial stem cells (SSCs). Similarly, pre- pubertal patients haven’t started spermatogenesis and cannot pro- vide sperm yet. When faced with gonadotoxic treatment for childhood malignancy, such as chemotherapy and radiation, they Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology, vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 27
- 41. may become azoospermic [3–5]. Consequently, current research efforts have focused on SSC therapy to treat male factor infertility. 28 Janmejay Hingu et al. Spermatogenesis begins with the SSC, a diploid cell that main- tains the ability for self-renewal and downstream differentiation into spermatozoa. Given its origin in spermatogenesis, using SSC for male infertility is a logical choice for patients with maturation arrest or initially diagnosed SCO with salvageable SSC on the biopsy [2, 6]. Investigative SSC therapies have included SSC trans- plantation and in vitro spermatogenesis. The formed differentiated germ cells could potentially be used for in vitro fertilization (IVF), either intracytoplasmic sperm injection (ICSI) or experimental round spermatid injection (ROSI) [7–10]. Given that SSC is a scarce population in azoospermic patients, in vitro propagation of SSC is a critical step toward the feasibility of stem cell-based fertility treatments. This chapter aims to provide our institution’s protocol and technical considerations for SSC culture in mammalians. Previous works from our lab have included SSC isolation, propagation, and partial differentiation to the round spermatid level in a unique culture system [10–12]. This was initially performed in adult men where SSC could be isolated and propagated for 28 weeks up to a concentration of 18,540-fold [11]. Subsequently, this application was expanded to prepubertal boys in which SSCs were cultured for up to 29 weeks [12]. Most recently, this culture system has been successfully applied to a mouse model and human patients with Klinefelter syndrome, given the frequency of azoospermia, negative mTESE, and the need for alternative therapies [8, 9, 13]. 2 Materials 2.1 Reagents 1. Phosphate-buffered saline (PBS, 1×). 2. Minimal Essential Media 1×. 3. Fetal calf serum. 4. DNase (Roche) stock solution of 2 mg/mL. It is stored at - 20 °C in 200 μL aliquots. Keep in ice before use. 5. Collagenase NB 4 (Serva) stock solution of 10 PZ U/mL. It is stored at -20 °C in 200 μL aliquots. Keep in ice before use. 6. Neutral Protease NB (Serva) stock solution of 10 DMC U/mL. It is stored at -20 °C in 100 μL aliquots. Keep in ice before use. 7. Enzyme mix: 5 mL 1× MEM DNase (8 μg/mL) + SERVA Collagenase 4 PZ U + neutral Protease* 0.2 DMC U + 20 μL extra DNase (2 mg/mL). If cells are intended for transplantation into humans, Neutral Protease NB GMP Grade is recommended.
- 42. Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells 29 8. Trypsin-EDTA 0.25% (Gibco). 9. 1 × MEM 10%FBS. 10. StemPro™ complete medium (Tables 1 and 2) (see Note 1). 2.2 Equipment 1. Laminar flow cabinet. 2. Shaking water bath with temperature control. 3. Water bath or bean bath with temperature control. 4. Centrifuge. 5. Microcentrifuge. 6. Pipette controller. 7. Serological pipettes (5, 10 mL). 8. Micropipettes. 9. Micropipette tips (2–1000 μL). 10. Conical tubes (10, 50 mL). 11. Microcentrifuge tubes (1.5 mL). 12. Nylon filter (77 μM, 55 μM). 13. Culture plates (size and surface will depend on the cells obtained). 14. Petri dishes. 15. Gloves. 16. Tweezers. 17. Surgical blade. 18. Aluminum foil. 19. Parafilm. 20. Ice. 21. Cell culture incubator. 22. Optical microscope. 23. Dissection microscope or stereoscope. 24. Hemocytometer or Bürker-Turk cell counting slide. 3 Methods 3.1 Before Isolation 1. Preheat water or bean bath to 37 °C. Fill with autoclaved water. Set culture media within to incubate before use. 2. Preheat the shaking water bath to 32 °C. Fill with autoclaved water. 3. Ensure sterile conditions are present inside the laminar flow cabinet.
- 43. Table 1 Chemicals used to supplement StemPro-34™ SFM – – – – – – - – – – – – – – – L 5 - – – L 1 – – – – L 30 nM L 60 – – 30 Janmejay Hingu et al. Chemicals/media Storage Stock conc. Solvent Amount for 500 mL Final conc. Solids Bovine albumin 4 °C 2.5 g 5 mg/mL D(+) glucose RT 3 g 6 mg/mL Ascorbic acid RT 8.8 mg 1 × 10 4 M Apo-Transferrin -20 °C 50 mg 100 μg/mL Pyruvic acid (sodium pyruvate) 4 °C 15 mg 30 mg/mL D-Biotin 4 °C 5 mg 10 μg/mL Liquids 2-betamercaptoethanol RT 1.7 μ × 10 5 M DL-lactic acid 4 °C 500 μ μL/mL MEM non-essential amino acids 4 °C 5 mL 10 μL/mL StemPro™-34 SFM Nutrient Supplement -20 °C 13 mL 26 μL/mL Insulin -20 °C 12.5 mg/mL 10 mM HCl 1 mL 25 μg/mL Sodium selenite -20 °C 0.25 mg/mL milliQ water 10 μ Putrescine -20 °C 100 mg/mL milliQ water 50 μ μM L-glutamine -20 °C 200 mM – 5 mL 2 mM MEM gitamin solution -20 °C 5 mL 10 μL/mL b-Estradiol -20 °C 0.6 mg/mL 100% EtOH 25 μL 30 ng/mL Progesterone -20 °C 0.6 mg/mL 100% EtOH 50 μL 60 ng/mL RT stands at room temperature
- 44. ™- di us ete Growth factors Storage (°C) Stock conc. Solvent Amount for 50 mL Final conc. Epidermal growth factor (EGF) -20 200 μg/mL 10 mM acetic acid/0.1% BSA 5 μL 20 ng/mL Human basic fibroblast growth factor (H bFGF) -20 10 μg/mL PBS/0.5%BSA 50 μL 10 ng/mL Leukemia inhibitor factor (LIF) 4 10 μg/mL – 50 μL 10 ng/mL Glial cell line-derived neurotrophic factor (GDNF) -20 10 μg/mL PBS/0.1% BSA 50 μL 10 ng/mL Fetal calf serum (FCS) -20 – – 0.5 mL 1% Penicillin/streptomycin (Pen/Strep) -20 – – 0.25 mL 0.5% Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells 31 Table 2 Growth factors added to supplemented StemPro 34 SFM imme ately before e to make StemPro™ compl
- 45. 32 Janmejay Hingu et al. 4. Ensure that the incubator has achieved optimal conditions for culture (37 °C, 5% CO2) and shows no signs of contamination. 5. The sample of testis tissue can be received from the operating room freshly or already cryopreserved viable tissue. The isola- tion process may vary slightly depending on whether fresh or frozen testis tissue is used. 6. Prepare 1× MEM with DNase mix (20 μL of 2 mg/mL DNase per 5 mL 1× MEM). Each sample will require about 100 mL. 7. Prepare 1× MEM 10% FBS (without DNase) for cell culture. 8. Prepare StemProTM Complete for cell culture (Tables 1 and 2) (see Note 2). 3.2 Isolation 1. Transfer approximately 20 mL of 1× MEM/DNase into three Petri dishes. 2. If the sample is fresh, deposit it on a petri dish with 1× MEM/- DNase and move on to step 5. If the tissue is frozen, allow it to adjust to room temperature for 2 min. Then continue thawing the cryovial with lukewarm running tap water and wipe off residual water with 70% ethanol (see Note 3). 3. As thawing continues, the tissue inside the cryotube will grad- ually transition from solid to liquid. When the tissue becomes liquid enough, but there is still a portion of solid pellet yet to be thawed, empty the cryovial contents into a petri dish contain- ing 1× MEM/DNase for the first wash. 4. Transfer the tissue into a second fresh petri dish containing 1× MEM/DNase using a pair of tweezers. Next, move the tissue into a new petri dish (the third one) containing 1× MEM/- DNase (see Note 4). 5. Remove the tunica albuginea, if necessary, using a pair of tweezers and a surgical blade (see Note 5). 6. Separate the seminiferous tubules of the testis apart and remove connective tissue, if present, using tweezers under a dissection microscope (Fig. 1). Note the tubules’ appearance (thick, thin, long, short, etc.) (see Note 6). Fig. 1 Bright-field microscopic view of seminiferous tubules before the addition of enzyme mix (a, b) and subsequent incubation in which the lumens of the tubules appear opaque (a, b) and transparent (c)
- 46. Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells 33 7. Transfer the tissue suspended in 1× MEM/DNase to a 15 mL tube. If tube contents appear clear, allow tubules to settle at the bottom of the tube. If tube contents appear turbid, centrifuge (5 min at 65 g without brake), and remove the supernatant. Wash with 1× MEM/DNase until the supernatant is clear. 8. Remove the supernatant, if present, from the tube until only 5 mL are left. Then add 5 mL of Enzyme Mix, bringing the total volume to 10 mL. 9. Close the tube and seal the cap with parafilm. Submerge the tube completely in the shaking water bath and incubate for 30 min (32 °C, 120 cycles/min). 10. Remove the tube from the incubator and open it. Without pipetting up and down, take a 10 μL sample and place it on a microscope slide for viewing (Fig. 1). If present, note the thickness of the peritubular cell layer and any fenestrations. Incubate longer, if necessary, until the tubules appear fene- strated, with most extra tubular cells released but still full of intratubular cells inside. 11. Centrifuge the tube (5 min at 16 g) without brake. Remove the supernatant if present. 12. Wash with 5 mL 1× MEM/DNase. Centrifuge (5 min at 16 g), without brake, and discard the supernatant. 13. Add 5 mL of fresh Enzyme Mix. 14. Close the tube and seal the cap with parafilm. Submerge the tube completely in the shaking water bath and incubate for 30 min (32 °C, 120 cycles/min). 15. Remove the tube from the incubator and open it. Pipette gently up and down about ten times. Take 10 μL and place it on a microscope slide for viewing (Fig. 1). Note changes in the peritubular cell layer thickness and fenestrations since the first incubation (see Note 7). 16. Discard any clumps of tubules, if present. 17. Filter the entire suspension through a 70 μM nylon filter into a new 50 mL tube. Then, filter the rest again through a 40 μM nylon filter into a new 50 mL tube. Cells of the exact origin may be pooled together during filtration. 18. Centrifuge (5 min at 350 g without brake). (a) If the supernatant is clear, discard it. (b) If the supernatant is turbid, take 10 μL and place it on a microscope slide for viewing. If large cells are present, repeat centrifugation to avoid losing stem cells.
- 47. 34 Janmejay Hingu et al. Fig. 2 Isolated testicular cells in culture. Seeded single cells after isolation (a), Germline Stem Cell cluster formation on top of somatic cells 3 weeks after cell culture (b, c) 19. Add 500 μL 1× MEM to the pellet and resuspend gently. Transfer to a microcentrifuge tube and spin down (5 min at 180 g, with brake). 20. Discard the supernatant and dissolve the pellet in ±500 μL 1× MEM/10% FCS (without DNase), depending on the size of the cell pellet. 21. Count the isolated cells using a Hemocytometer. 22. Culture cells in a 6-well plate with 1× MEM/10% FBS at a concentration of 5000–10,000 cells/cm2 . Incubate overnight at 37 °C, 5% CO2. 3.3 Cell Culture 1. Keep the cells inside the incubate at 37 °C, 5% CO2, replacing 1× MEM/10% FBS with StemPro complete medium (Tables 1 and 2). 2. Observe the cells in the culture regularly (Fig. 2). Note cell morphology, the confluence of attached cells, and cell debris or contamination (see Notes 8 and 9). 3. Refresh culture media 2–3 times a week. Use the media volume recommended by the manufacturer. Avoid losing floating cells by collecting spent media in a tube and centrifuging to form a pellet. Resuspend the pellet with new media and reintroduce the volume evenly among culture wells. Shake the plate gently to ensure the cells seeded spread evenly around the well (see Note 10). 4. When 80–90% confluency is achieved, cells should be trypsi- nized and passage into a new plate to continue propagation. If somatic cell overgrowth is suspected, differential plating can be considered 12–48 h after seeding. 3.4 Cell Cryopreservation 1. Passage cells with trypsin EDTA incubation and then count them. 2. Plan the way you want to freeze your cells depending on the study’s goals and cell availability (see Note 11).
- 48. Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells 35 3. Centrifuge cells (5 min, 180 g, with break). Remove the super- natant and resuspend the pellet in 750 μL 1× MEM/20% FCS (Room Temperature). 4. Transfer the cell suspension into a cryovial. 5. Add 750 μL 1× MEM/20% FCS/16% DMSO while gently shaking the cryovial. 6. Transfer the cryovial into a cold Nalgene freezing box (Mr. Frosty). Maintain at -80 °C for around 12 h but no longer than 24 h. 7. Transfer the cryovial to a liquid N2 tank. 4 Notes 1. The reagents shown in Table 1 are added to 500 mL StemPro™-34 serum-free medium to supplement the medium. Final concentrations are based on the 500 mL StemPro™-34 SFM, not the total volume. 2. After mixing reagents, filter the mixture with vacuum pressure over a 22 μM filter. Immediately before use, add growth factors shown in Table 2 to 50 mL supplemented StemPro™ SFM. 3. An aggressive change in the temperature of the cryovial may trigger an expansive reaction of the gas inside. This may break the cryovials, compromising the sample integrity or even becoming a safety hazard for the operator. Ensure the initial thawing is gradual and that appropriate safety guideline is followed. We also recommend briefly opening the cryovials to release air pressure as soon as the thawing process allows. 4. As previously reported, dimethyl sulfoxide DMSO is one of the most commonly used cryoprotectants for testicular tissue [14], but at room temperature, DMSO becomes cytotoxic. Three time-sensitive consecutive washes are done to minimize tissue exposure to DMSO at room temperature. 5. During this step, it is recommended to weigh the tissue to calculate the isolation efficacy (number of cells per mg tissue). 6. Seminiferous tubules from simple testicular biopsies are usually more laborious to separate than in mTESE samples. The further separated the tubules are, the more adherent (mucous-like) they appear and the more influential the enzy- matic digestion will be. 7. The tubular cell layer should disintegrate at this point, releasing the stem cells. The thickness of the tubular cell layer can differ between patients. The incubation with enzymes can be continued for longer to achieve the desired characteristics of the peritubular cell layer. If cell clumps form, dissociate them with trypsin-EDTA.
- 49. 8. 36 Janmejay Hingu et al. 8. Taking pictures of the cells in culture is encouraged. 9. If there is uncertainty about cell viability, Live/Dead staining (Invitrogen LIVE/DEAD™ Viability/Cytotoxicity Kit) fol- lowed by fluorescence microscopy can be performed before refreshing media. 10. Avoid keeping cultured cells without media for too long dur- ing the refresh process. 11. In our experience, we use 1.8 mL cryovials with up to 4,000,000 cells per cryovial. Higher cell concentrations may compromise cell viability after thawing. References 1. Zegers-Hochschild F et al (2017) The interna- tional glossary on infertility and fertility care, 2017. Hum Reprod 32(9):1786–1801 2. Abdelaal NE et al (2021) Cellular therapy via spermatogonial stem cells for treating impaired spermatogenesis, non-obstructive azoosper- mia. Cell 10(7):1779 3. Halpern JA et al (2020) Oncofertility in adult and pediatric populations: options and barriers. Transl Androl Urol 9(Suppl 2):S227–S238 4. David S, Orwig KE (2020) Spermatogonial stem cell culture in oncofertility. Urol Clin North Am 47(2):227–244 5. Diao L et al (2022) Roles of spermatogonial stem cells in spermatogenesis and fertility res- toration. Front Endocrinol (Lausanne) 13: 895528 6. Xi HM et al (2022) Recent advances in isola- tion, identification, and culture of mammalian spermatogonial stem cells. Asian J Androl 24(1):5–14 7. Tanaka A et al (2018) Ninety babies born after round spermatid injection into oocytes: survey of their development from fertilization to 2 years of age. Fertil Steril 110(3):443–451 Galdon G et al (2021) In vitro propagation of XXY undifferentiated mouse spermatogonia: model for fertility preservation in Klinefelter syndrome patients. Int J Mol Sci 23(1):173 9. Galdon G et al (2022) In vitro propagation of XXY human Klinefelter spermatogonial stem cells: a step towards new fertility opportunities. Front Endocrinol (Lausanne) 13:1002279 10. Pendergraft SS et al (2017) Three-dimensional testicular organoid: a novel tool for the study of human spermatogenesis and gonadotoxicity in vitro. Biol Reprod 96(3):720–732 11. Sadri-Ardekani H et al (2009) Propagation of human spermatogonial stem cells in vitro. JAMA 302(19):2127–2134 12. Sadri-Ardekani H et al (2011) In vitro propa- gation of human prepubertal spermatogonial stem cells. JAMA 305(23):2416–2418 13. Deebel NA et al (2022) Morphometric and immunohistochemical analysis as a method to identify undifferentiated spermatogonial cells in adult subjects with Klinefelter syndrome: a cohort study. Fertil Steril 118(5):864–873 14. Zarandi NP et al (2018) Cryostorage of imma- ture and mature human testis tissue to preserve spermatogonial stem cells (SSCs): a systematic review of current experiences toward clinical applications. Stem Cells Cloning 11:23–38
- 50. Chapter 4 Purification by STA-PUT Technique of Male Germ Cells from Single Mouse and RNA-Extraction for Transcriptomic Analysis Chiara Naro, Claudio Sette, and Raffaele Geremia Abstract Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcrip- tional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post- meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals. Key words Male germ cells, Cell purification, STA-PUT, Gravimetric sedimentation, RNA extraction, Transcriptome analysis 1 Introduction Spermatogenesis is the complex differentiation process taking place in the testes, leading to the generation of mature male gametes [1, 2]. During this process spermatogonial stem cells undergo profound genomic and morphological variations, which occur along three subsequential phases: (i) amplification of spermatogo- nia population, (ii) meiotic commitment and division into primary and secondary spermatocytes, (iii) differentiation from haploid round spermatids to mature spermatozoa (i.e. spermiogenesis) [1, 2]. Initiation and accomplishment of each of these phases of spermatogenesis is sustained by stage-specific patterns of gene expression, providing each phase of the necessary protein repertoire [3–7]. Understanding of the biological processes and of underlying molecular mechanisms driving spermatogenesis relies on the Marco Barchi and Massimo De Felici (eds.), Germ Cell Development: Methods and Protocols, Methods in Molecular Biology, vol. 2770, https://doi.org/10.1007/978-1-0716-3698-5_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 37
- 51. possibility to isolate and analyze male germ cells at the defined stage of differentiation. In this regard, the STA-PUT technique repre- sents, since the late 1960s, a pivotal procedure in this field of study. This technique takes advantage of the fact that the individual types of testicular cells have different size, thus sedimenting at different velocities in a BSA gradient, independently from the shape. The physical characteristics of the method have been extensively described by Miller and Phillips using blood cells [8] and then used for the separation of mouse male germ cells in the laboratory of Dr. W.R. Bruce [9]. The effectiveness of the technique has been re-evaluated from a morphological point of view [10], re-examined also recently proposing different equipment [11], and widely used for the study of DNA synthesis and transcriptional and translational activity in the different phases of spermatogenesis and for a variety of biochemical and metabolic studies [12–16]. The recent advance- ment of transcriptomic approaches has provided remarkable insights into the transcriptional and post-transcriptional molecular mechanisms underlying the tightly and dynamically controlled gene programs promoting male germ cells differentiation [17, 18]. Most of these studies exploited RNA-sequencing (RNA-seq) from either bulk-testis, collected at different time-points during the first syn- chronous wave of murine spermatogenesis, or from male germ cells at different stage of differentiation, isolated by either centrifugal elutriation or Fluorescence-activated Cell Sorting (FACS) [3, 6, 7]. Both these approaches show limitations: i. presence of hetero- geneous populations of somatic cells (i.e., Sertoli cells, Leydig cells) is a confounding element in the identification of germ-cell-specific gene expression patterns by RNA-seq of bulk-testes; ii. stage- specific male germ cell isolation by centrifugal elutriation requires injection of a high number of testicular cells obtained by pooling testes from multiple mice. This latter aspect is a critical limitation for transcriptomic studies aimed at the identification of gene expression alterations correlated to spermatogenic defects in mice with reduced reproductive potential, due to the small number of animals. In our laboratory, we showed that gravimetric decantation (STA-PUT) of testicular cellular suspension is a reliable method to collect homogenous population of meiotic (spermatocytes) and post-meiotic (round spermatids) male germ cells from a single mouse, from which it is possible to isolate a sufficient amount of RNA for reliable total-RNA seq analysis [4]. Moreover, we showed this method to be applicable and reliable also for the isolation of germ cells and subsequent transcriptomic analysis from a mouse model with relevant spermatogenic defects, with reduced testis size and impaired male germ cells differentiation, such as the Sam68 knock-out model [19, 20], therefore proving its applicability to the late-generation transcriptomic analysis. Herein, we will describe a detailed protocol for the fractionation of meiotic spermatocytes and post-meiotic round spermatids from a single adult mouse and the isolation of good-quality RNA for further transcriptomic analysis. 38 Chiara Naro et al.
- 52. Male Germ Cells Isolation from Single Mouse Using STA-PUT 39 2 Materials 2.1 Animals The procedure described in this chapter refers to testicular germ cells suspension obtained from the digestion of testes of 12-week old mouse from the C57BL/6 strain (see Note 1). 2.2 BSA Gradients 1. 1.5 L EKRB (120.1 mM NaCl, 4.8 mM KCl, 25.2 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2, 11 mM). All components are diluted in water. This solution is used for the preparation of both the BSA gradients and the testicular germ cells suspension. Solution is filtered through a 0.22 μM filter and stored at 4 °C (see Note 2). 2. Bovine serum albumin (BSA) 3% in EKRB: Dissolve 12 g BSA in 400 mL of EKRB at room temperature using magnetic stirring. The solution is filtered through a 0.22 μM filter and stored at 4 °C. 250 mL and will be used as such to create the gradient. The excess will be used to prepare 250 mL of 1% BSA in EKRB, 60 mL 0.5% BSA in EKRB, and 15 mL 0.2% BSA in EKRB by the dilution of an appropriate amount of EKRB w/o BSA (see Notes 3 and 4). 2.3 STA-PUT Apparatus 1. Sedimentation chamber: The sedimentation chamber had a conical base carved out of a square plate with a side of 15 cm and a thickness of 4 cm. The cone had a diameter of 12 cm at the base and was 3 cm deep with a hole of 4 mm diameter at the apex. Above the conical base a 15 cm long tube with an internal diameter of 12 cm was glued to complete the chamber. 2. Three cylinders, necessary for the formation of the albumin gradient to be loaded into the sedimentation chamber, with the following characteristics: (a) Two cylinders measuring 7.5 cm internal diameter and 15 cm height on a square base with 10 cm sides to ensure stability. At the base of one cylinder (n.1) there is one outlet represented by a small conical plexiglass tube with an internal diameter of 2 mm; at the base of the second cylinder (n.2), there are two of such outlets placed at the two ends of the cylinder diameter. (b) The third cylinder measured 1.8 cm internal diameter and 15 cm height on a square base of 5 cm sides to ensure stability. At the base of the cylinder (n.3), there are two outlets as described for cylinder n.2 (see Note 5). 3. One baffle: A 1.2 cm diameter stainless steel hemisphere with three 2 mm pegs (see Note 6). 4. Silicone tubing 2 mm internal diameter.
- 53. 40 Chiara Naro et al. 5. Straight mini tubing connector to fix the tubing to the base hole of the STA-PUT. 6. One 10 mL syringe for sample injection, hold by a support clamp, attached through a bosshead to a laboratory stand. 7. Two 3-way stopcocks and luer lock adapters if necessary to connect tubing. 8. Three curved tubing clamps, placed on the connecting tubes between the cylinders and the syringe. 9. One tubing clip, Hoffman pattern, placed on the connecting tube between the syringe for sample injection and the STA- PUT chamber. 10. Two magnetic stirrers for cylinders n. 2 and 3. 11. Two magnetic stirring bars for cylinders n. 2 and 3. 12. A small laboratory jack to support the cylinder n.1 at the same level of cylinders 2 and 3. 13. A perforated support about 10 cm high for the STA-PUT (laboratory jack or polystyrene box). 2.4 Testicular Germ Cells Preparation 1. Shaking water bath set at 32 °C. 2. Refrigerated bench centrifuge. 3. Sterile blades. 4. 15 mL sterile polypropylene tubes. 5. Hemocytometer for cell counting. 6. Minimum Essential Medium (MEM). 7. Collagenase from Clostridium histolyticum (Sigma Aldrich, Cat # C7657; CAS 9001-12-1): 5 mg/mL in MEM, equal to a 20-fold stock solution (see Note 7). 8. DNase I from bovine pancreas (Sigma Aldrich, Cat # DN25; CAS 9003-98-9): 2 mg/mL dissolved in MEM, equal to a 40-fold stock solution (see Note 7). 9. Trypsin from bovine pancreas (Sigma Aldrich Cat # T9201; CAS 9002-07-7): 10 mg/mL in MEM, equal to a ten-fold stock solution (see Note 7). 10. Fetal bovin serum (FBS). 2.5 Fractions Collection and Analysis of Germ Cells Nuclear Morphology 1. Refrigerated bench centrifuge. 2. 15 and 50 mL sterile polypropylene tubes. 3. 1.5 mL sterile polypropylene tubes. 4. Hoechst 33342 10 mg/mL in water (ThermoFisher Scientific Cat #H3570). 5. Microscope slides.
- 54. Male Germ Cells Isolation from Single Mouse Using STA-PUT 41 2.6 RNA Purification 1. Refrigerated bench centrifuge. 2. miRNeasy Mini Kit with DNase (Qiagen). 3. DNAse-RNAse free 1.5 mL tubes and tips (see Note 8). 3 Methods 3.1 Assembly of the STA-PUT Apparatus Prior or during the germ cells isolation procedure, place the appa- ratus in a cold room or in a large refrigerator, where all procedures will be performed. The setup of the apparatus is illustrated in Fig. 1. 1. Place Cylinders n. 2 and n. 3, with a stirring bar of the proper size inside, on two magnetic stirrers. Place the cylinder n. 1 on a laboratory jack of the same height as the stirrers. The three cylinders should be connected with silicone tubing and placed on a shelf approximately 30 cm above the base of the STA-PUT to allow for gradient flow during loading. 2. Place firmly the STA-PUT chamber, with the baffle inside (see Note 9), on the perforated support to allow the passage of the cell suspension and the gradient to and from the base hole of the chamber. Fig. 1 The STA-PUT apparatus: (a) cylinder n.1 containing 3% BSA; (b) cylinder 2 containing 1%BSA; (c) cylinder 3 containing 5% BSA; (d) 10 mL syringe for EKRB and cell suspension loading; (e) silicone tubing; (f) magnetic stirring bars; (g) curved tubing clamps; (h) three-way stopcock; (i) small laboratory jack; (l) magnetic stirrer; (m) laboratory stand; (n) support clamp and boss-head; (o) tubing clips, Hoffman pattern; (p) perforated laboratory jack; (q) STA-PUT; (r) baffle; (s) straight mini tubing connector; (t) exit for the collection of fractions
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- 56. at .28. Sold to J. C. Peters Son, 267 Roberts St., 3 chests Oolong tea, 146# at .52, 5 sacks Rio coffee, 252# at .48. Bought for cash from Harris Co., 1 office desk and chair $45.00, gave Ck. No. 3 in payment. —4th— Paid freight on coffee from New York by Ck. No. 4, 12.93. Sold to Wright Noble, 146 7th St., 2 sacks Java coffee, 99# at .32; 2 sacks Mocha coffee, 101# at .32. Sold to Horgis Co., 84 Jackson St., 5 chests Japan tea, 248# at .48. Sold to Winters James, 92 Hastings St., 4 chests Japan tea, 201# at .48; 3 chests Oolong tea, 138# at .52; 2 sacks Java coffee, 97# at .32. Sold for cash 1 sack Rio coffee, 47# at .28; 1 chest Japan tea, 45# at .48. —5th— Sold to Cobb Willet, 892 Park Av., 2 chests Japan tea, 92# at .48; 1 chest Oolong tea, 47# at .52; 1 sack Rio coffee, 44# at .28; 1 sack Java coffee, 45# at .32; 1 sack Mocha coffee, 43# at .32. Sold to Young Criger, 62 Watson St., 5 sacks Mocha coffee, 205# at .32; 3 chests Oolong tea, 127# at .52. Bought from Japan Importing Co., San Francisco, 60 chests Japan tea, 2,700# at .36, f. o. b. Omaha, net cash; gave our note at 10 days without interest in payment. Paid account of Leggitt Co., less 2% discount, Ck. No. 5. —6th— Ames Johnson paid their account, less 2% cash discount. Deposited cash received to date. Sold to Wade Francis, 92 Bluff St., 10 chests Japan tea, 448# at .48. Paid for telegram—petty cash—.40.
- 57. Received check from Landis Snow in full settlement of their account. Sold to J. C. Peters Son, 5 sacks Java coffee, 231# at .32. Sold for cash 3 sacks Rio coffee, 127# at .28; 2 sacks Mocha coffee, 89# at .32; 3 sacks Japan tea, 131# at .48. —8th— Deposited cash on hand, also check of Landis Snow. Sold to Ames Johnson, 2 sacks Mocha coffee, 91# at .32; 2 chests Oolong tea, 87# at .52. Sold to Wright Noble, 3 chests Japan tea, 129# at .48; 1 chest Oolong tea, 42# at .52. Paid for fuel by check No. 6 to Rogers Coal Co., 12.00. Paid clerk's salary, check No. 7, 10.00. Paid for labor, check No. 8, 16.50. —9th— Sold to Watkins Fish, 64 Prairie Av., 5 chests Oolong tea, 207# at .52. Bought from Western Grocer Co., Chicago, 50 chests Oolong tea, 418# at .39; 20 sacks Rio coffee, 876# at .22¼; 10 sacks Java coffee, 434# at .25; 15 sacks Mocha coffee, 653# at .25; terms 30 days net, 2/10, f. o. b. Omaha. Received from Wright Noble cash in payment of our bill of Jan. 4th, less 2% cash discount. —10th— Received from bank, check of Landis Snow protested for non- payment, protest fees added 1.90. Sent Laughlin Co. our check No. 9 in payment of account —11th— Sold to Raymond H. Moss, 182 Spring St., 5 chests Japan tea, 217# at .48; 5 sacks Rio coffee, 214# at .28.
- 58. Sold to Watkins Fish, 10 sacks Mocha coffee, 424# at .32. Cobb Willet paid their account in full, deducting 2% for cash. —12th— Deposited cash on hand. Sold to Cobb Willet, 5 chests Japan tea, 213# at .48. Sold for cash, 2 sacks Rio coffee, 88# at .28. —13th— Paid our note to Japan Importing Co., check No. 10. Paid sundry office expenses from petty cash 3.60. Sold to Wade Francis 3 sacks Rio coffee, 123# at .28; 2 sacks Mocha coffee, 86# at .32. Paid clerk's salary, ck. No. 11, 10.00. Paid for labor, ck. No. 12, 16.50. —15th— Paid ½ month's salary to D. C. Hoadley, ck. No. 13, 75.00. 1st. Balance cash, first charging petty cash expenditures as a cash payment. 2nd. Post purchase book, sales book, journal, and cash book. 3rd. Take a trial balance. 4th. Credit interest to partner's accounts. 5th. Take an inventory of stock on hand. The records show quantities purchased and quantities sold. When the same goods have been purchased at different prices, use the last price paid in figuring inventory. 6th. Close accounts into trading and profit and loss accounts. 7th. Distribute net profits to partners' capital accounts.
- 59. COMBINED ORDER AND SALES RECORD 17. Instead of keeping separate order and sales books, both records may be combined on one blank. This is accomplished by the use of order blanks provided with columns for the distribution of sales to the different departments. Before the order is filled these blanks are handled in the same manner as those without distribution columns. When filled, the amounts are extended in the proper columns and the invoice made. The orders are then filed in a binder, each day's orders being kept together, and postings made direct to customers' accounts. The footings are carried forward to the end of the month and totals posted to sales accounts. The original orders thus become a loose leaf sales book.
- 60. ABSTRACT OF SALES 18. When the order blank is used as a sales record, making a sales book with a record of a single sale to a sheet, it is somewhat inconvenient to determine from the footings the total sales for the day. This information is of considerable value, as a knowledge of what is being done from day to day is of importance to the principals of a business. Such a record is provided for by an abstract of sales on a separate sheet. This abstract should show total sales for each day, both cash and on account, divided by departments. The blank may be made the same size as the order blanks, and filed in the sales binder at the beginning of the month. Sales are recorded daily and footings carried forward to the end of the month, when the totals may be posted direct to the credit of the sales account in the general ledger. The totals of sales on account will be posted to the debit of the sales controlling account in the general ledger.
- 61. OUT FREIGHT 19. The proper treatment of freight paid on outgoing shipments is an important question in accounting. When goods are sold at delivered prices, the freight paid is one of the items of expense in selling the goods, and when the books are closed the account will be closed into profit and loss. However, in a wholesale business, freight is sometimes paid as an accommodation to the customer when the goods have been sold at f. o. b. prices. Although the amount should be added to the invoice it should not be credited to the sales account as this would be taking credit for a fictitious trading profit. Such an item should be made a special charge against the customer by means of a journal entry. Customer Dr. Out Freight Cr.
- 62. Combined Order and Sales Record
- 63. THE GENERAL OFFICES OF THE SAMUEL C. TATUM CO., CINCINNATI, OHIO
- 64. Abstract of Sales by Departments
- 65. SALES EXPENSE 20. In a wholesale or manufacturing business it is very desirable that the exact cost of selling goods be known. Broadly, this cost is covered under the general head of sales expense, but this is usually divided into several classes of expenditures. The segregation of the various items of sales expense is desirable for the purpose of determining the percentage of each. The items which properly belong in sales expense depend somewhat on the nature of the business. For example, traveling expenses are usually a direct sales expense, but in some businesses they may be chargeable to the cost of purchases. The items entering into sales expense of the average business are: advertising, salaries of salesmen, traveling expenses of salesmen, commissions paid on sales, cost of packing and shipping, out freight. 21. Advertising. This account should be charged with all expenditures for publicity such as newspaper, magazine, street car, and bill board advertising, cost of printing catalogs, booklets, and circulars. Where there is any reason for so doing, the cost of the different classes of advertising can, of course, be kept in separate accounts. The aim and object of advertising being to increase the sale of goods, it is properly considered an item of sales expense. 22. Salaries of Salesmen. This account is charged with all salaries paid to salesmen whether traveling or working in the house. Commissions and bonuses are sometimes included in this account, but it is usually considered best to keep them in separate accounts. 23. Traveling Expenses of Salesmen. This account is charged with all legitimate traveling expenses of salesmen, the specific items included depending largely on the nature of the business. For example, in some businesses a liberal allowance is made for the entertainment of customers, while in others this item is never allowed. In any business the traveling expense account requires careful scrutiny. Salesmen should be required to furnish an itemized statement or voucher of expenses at stated intervals. For convenience, this should be made on a form specially provided for the purpose. One of the most convenient and popular expense vouchers is in the form of a book of a convenient pocket size, with a page for each day of the week and a summary of the week's expenses on the last page.
- 66. 24. Packing and Shipping. This account is charged with the entire cost of packing goods for shipment. It includes such items as wages of shipping clerk and his assistants, crates, lumber, boxes, and all other packing materials.
- 67. TRIAL BALANCE BOOK Traveler's Expense Book 25. To save rewriting the names of the accounts each month, a trial balance book can be used to good advantage. These books are made to
- 68. accommodate six trial balances on a double page, and are sometimes made with alternate short leaves so that twelve trial balances may be made with one writing of the names. When the trial balance book is used, care must be exercised in providing space for the addition of new accounts in each section. Where separate sales and purchase ledgers are used, it is best to provide a trial balance book for each ledger.
- 69. THE CHECK REGISTER 26. Large check books are cumbersome to handle and necessitating the expenditure of much needless labor. Their use is rapidly giving way in modern offices to the check register. The check register has several distinct advantages. It exhibits, in compact form, a record of all checks issued and can also be arranged to show deposits and balance in the bank. Distribution columns can be provided with headings for the different expenditure accounts, which makes of the check register a cash expenditure book. The form should be varied to suit the business in which it is to be used. A typical form is illustrated on page 37. 27. Checks in Pads. When the check register is used it is the usual custom to have checks put up in pads. After the check is written, it is registered and numbered to correspond to the register number. With the use of padded checks, it is not necessary for the clerk who writes the check to know anything about the bank balance. Trial Balance Book
- 70. Check Register Combined with Cash Expenditure Book Cash Received Book
- 71. Checks in Pads
- 72. CASH RECEIVED BOOK 28. A cash book specially ruled for a record of cash received is used to supplement the check register or cash expenditure book. Columns are provided for the different classes of receipts, with one credit column. It is assumed that all cash received is deposited, payments being made exclusively by check. This does not refer to petty cash expenditures which should be kept in a petty cash book or on envelope vouchers.
- 73. SAMPLE TRANSACTIONS 29. The following transactions illustrate the use of the special blanks and books described. D. A. Hall employs H. D. Snyder as traveling salesman for the purpose of increasing his business, agreeing to pay him a salary of $150.00 per month and expenses. He commences work on Feb. 11th. The amounts in the ledgers stand as shown in the last model set illustrated and these transactions are recorded: —Feb. 11th.— Paid Altman Sons To balance account Ck. No. 9 $350.00 —11th— Paid Garson Co. To balance account Ck. No. 10 175.00 —11th— Sold to Daniels Dean, Boone, Ia. 10 men's suits $7.50 $75.00 10 men's suits 6.75 67.50 20 boys' suits 2.00 40.00 182.50 Terms 2/10 N/30 —11th— Sold to A. C. Petersen, Nevada, Ia. 10 men's overcoats 8.50 85.00
- 74. 10 men's suits 7.00 70.00 155.00 Terms 2/10 N/30 —11th— Received from D. A. Marcus Son Cash 139.65 Discount 2% 2.85 —11th— Sold for cash 20 boys' suits 1.75 35.00 20 men's pants 2.00 40.00 —12th— Received from John Gorham Cash 132.30 Discount 2% 2.70 —12th— Sold to Henry Cook, Iowa Falls, Ia. 5 men's suits 8.00 40.00 5 men's suits 5.75 28.75 5 boys' suits 2.00 10.00 5 boys' suits 1.75 8.75 87.50 Terms 2/10, N/30 —12th— Sold to James Adams, Dennison, Ia. 15 men's suits 7.00 105.00 Terms 2/10, N/30
- 75. —12th— Received from Geo. Golden Cash $150.00 —13th— Sold to D. A. Marcus Sons 10 men's pants 2.00 20.00 10 boys' suits 1.75 17.50 37.50 Terms 2/10, N/30 —13th— Paid freight on shipment to Henry Cook Ck. No. 11 3.65 Charge to Cook —13th— Deposited cash 824.45 —13th— Sold to S. H. Allen, Mason City, Ia. 30 men's pants 1.75 52.50 Terms 2/10, N/20 —14th— Paid Adler Co. Ck. No. 12 303.12 Discount 9.38 —15th— Sold to Marx Sons, Charles City, Ia.
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